Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin

ABSTRACT

A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/863,772, filed Aug. 26, 2010, which is a national phase applicationof PCT/CA2009/000032, filed Jan. 12, 2009, which claims priority to U.S.Provisional Application No. 61/022,775, filed Jan. 22, 2008, andCanadian Application No. 2,615,372, filed Jan. 21, 2008;PCT/CA2009/000032 is also a Continuation-In-Part of and claims priorityfrom PCT Application No. PCT/CA2008/001281, filed Jul. 11, 2008.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 267342001110SequenceListing.txt, date recorded: Jan. 9, 2013, size: 304 KB).

FIELD OF INVENTION

The present invention relates to the production of virus-like particles.More specifically, the present invention is directed to the productionof virus-like particles comprising influenza antigens.

BACKGROUND OF THE INVENTION

Influenza is the leading cause of death in humans due to a respiratoryvirus. Common symptoms include fever, sore throat, shortness of breath,and muscle soreness, among others. During flu season, influenza virusesinfect 10-20% of the population worldwide, leading to 250-500,000 deathsannually.

Influenza viruses are enveloped viruses that bud from the plasmamembrane of infected mammalian and avian cells. They are classified intotypes A, B, or C, based on the nucleoproteins and matrix proteinantigens present. Influenza type A viruses may be further divided intosubtypes according to the combination of hemagglutinin (HA) andneuraminidase (NA) surface glycoproteins presented. HA governs theability of the virus to bind to and penetrate the host cell. NA removesterminal sialic acid residues from glycan chains on host cell and viralsurface proteins, which prevents viral aggregation and facilitates virusmobility. Currently, 16 HA (H1-H16) and 9 NA (N1-N9) subtypes arerecognized. Each type A influenza virus presents one type of HA and onetype of NA glycoprotein. Generally, each subtype exhibits speciesspecificity; for example, all HA and NA subtypes are known to infectbirds, while only subtypes H1, H2, H3, H5, H7, H9, H10, N1, N2, N3 andN7 have been shown to infect humans (Horimoto 2006; Suzuki 2005).Influenza viruses comprising H5, H7 and H9 are considered the mosthighly pathogenic forms of influenza A viruses, and are most likely tocause future pandemics.

Influenza pandemics are usually caused by highly transmittable andvirulent influenza viruses, and can lead to elevated levels of illnessand death globally. The emergence of new influenza A subtypes resultedin 4 major pandemics in the 20^(th) century. The Spanish flu, caused byan H1N1 virus, in 1918-1919 led to the deaths of over 50 million peopleworldwide between 1917 and 1920. Presently, the risk of the emergence ofa new subtype, or of the transmission to humans of a subtype endemic inanimals, is always present. Of particular concern is a highly virulentform of avian influenza (also called “bird flu”), outbreaks of whichhave been reported in several countries around the world. In many cases,this bird flu can result in mortality rates approaching 100% within 48hours. The spread of the avian influenza virus (H5N1), first identifiedin Hong Kong in 1997, to other Asian countries and Europe has beenpostulated to be linked to the migratory patterns of wild birds.

The current method of combating influenza in humans is by annualvaccination. The vaccine is usually a combination of several strainsthat are predicted to be the dominant strains for the coming“flu-season”. The prediction is coordinated by the World HealthOrganization. Generally, the number of vaccine doses produced each yearis not sufficient to vaccinate the world's population. For example,Canada and the United-States obtain enough vaccines doses to immunizeabout one third of their population, while only 17% of the population ofthe European Union can be vaccinated. It is evident that currentworldwide production of influenza vaccine would be insufficient in theface of a worldwide flu pandemic. Even if the necessary annualproduction could somehow be met in a given year, the dominant strainschange from year to year, thus stockpiling at low-need times in the yearis not practical. Economical, large scale production of an effectiveinfluenza vaccine is of significant interest to government and privateindustry alike.

The viral stocks for use in vaccines are produced in fertilized eggs.The virus particles are harvested, and for an inactivated viral vaccine,disrupted by detergent to inactivate. Live attenuated vaccines are madeof influenza viruses that were adapted for growth at low temperaturewhich means that at normal body temperature, the vaccine is attenuated.Such a vaccine is licensed in USA for use in individuals from 5 to 49years of age. Inactivated whole virus vaccines are rendered harmless byinactivation with chemical agents and they have been produced inembryonic eggs or mammalian cell culture. All these types of vaccineshow some specific advantages and disadvantages. One advantage ofvaccines derived from whole viruses is the type of immunity induced bysuch vaccines. In general, split vaccines induce a strong antibodyresponse while vaccines made of whole viruses induce both an antibody(humoral) and cellular response. Even though a functional antibodyresponse is a criterion for licensure that correlates with protectioninduced by a vaccine, there is increasing evidence that a T-cellresponse is also important in influenza immunity—this may also providebetter protection in the elderly.

In order to induce a cellular immune response, vaccines made of wholeviruses were developed. Due to the high pathogenicity of the influenzastrain (e.g. H5N1), these vaccines are produced in BL3+ facility. Forhighly pathogenic influenza strains such as H5N1, some manufacturershave modified the hemagglutinin gene sequence in order to reduce thepathogenicity of the influenza strain and to make it avirulent and moreeasily produced in embryonic eggs or mammalian cell culture. Others alsouse reassortant influenza strains in which the genetic sequences for thehemagglutinin and neuraminidase proteins are cloned in a high-yieldinglow pathogenic influenza donor strain (A/PR/8/34; Quan F-S et al, 2007).While these methods may produce useful vaccines, they do not provide asolution to the need for high-volume, low cost and fast production ofvaccines in the scale necessary to meet the global need in a normalyear, and would almost certainly be insufficient in the face of apandemic.

Using this reverse genetic technology, one might also need to mutate thegenetic sequence of the HA protein to make it avirulent. For highlypathogenic influenza strains, the production of whole virus vaccineseither requires confinement procedures or the resulting vaccines do notexactly match the genetic sequence of the circulating virus. In the caseof live-attenuated vaccines, there is still a risk that the administeredvaccine can recombine with an influenza virus from the host, leading toa new influenza virus.

While this method maintains the antigenic epitope and post-translationalmodifications, there are a number of drawbacks to this method, includingthe risk of contamination due to the use of whole virus and variableyields depending on virus strain. Sub-optimal levels of protection mayresult from genetic heterogeneity in the virus due to its introductioninto eggs. Other disadvantages includes extensive planning for obtainingeggs, contamination risks due to chemicals used in purification, andlong production times. Also, persons hypersensitive to egg proteins maynot be eligible candidates for receiving the vaccine.

In the case of a pandemic, split vaccine production is limited by theneed to adapt the strain for growth in eggs and the variable productionyields achieved. Although this technology has been used for years forthe production of seasonal vaccines, it can hardly respond in areasonable timeframe to a pandemic and worldwide manufacturing capacityis limited.

To avoid the use of eggs, influenza viruses have also been produced inmammalian cell culture, for example in MDCK or PERC.6 cells, or thelike. Another approach is reverse genetics, in which viruses areproduced by cell transformation with viral genes. These methods,however, also requires the use of whole virus as well as elaboratemethods and specific culture environments.

Several recombinant products have been developed as recombinantinfluenza vaccine candidates. These approaches have focused on theexpression, production, and purification of influenza type A HA and NAproteins, including expression of these proteins using baculovirusinfected insect cells (Crawford et al, 1999; Johansson, 1999), viralvectors, and DNA vaccine constructs (Olsen et al., 1997).

Specifics of an influenza virus infection are well known. Briefly, theinfectious cycle is initiated by the attachment of the virion surface HAprotein to a sialic acid-containing cellular receptor (glycoproteins andglycolipids). The NA protein mediates processing of the sialic acidreceptor, and virus penetration into the cell depends on HA-dependentreceptor-mediated endocytosis. In the acidic confines of internalizedendosomes containing an influenza virion, the HA protein undergoesconformational changes that lead to fusion of viral and cell membranesand virus uncoating and M2-mediated release of MI proteins fromnucleocapsid-associated ribonucleoproteins (RNPs), which migrate intothe cell nucleus for viral RNA synthesis. Antibodies to HA proteinsprevent virus infection by neutralizing virus infectivity, whereasantibodies to NA proteins mediate their effect on the early steps ofviral replication.

Crawford et al. (1999) disclose expression of influenza HA inbaculovirus infected insect cells. The expressed proteins are describedas being capable of preventing lethal influenza disease caused by avianH5 and H7 influenza subtypes. Johansson et al. (1999) teach thatbaculovirus-expressed influenza HA and NA proteins induce immuneresponses in animals superior to those induced by a conventionalvaccine. Immunogenicity and efficacy of baculovirus-expressedhemagglutinin of equine influenza virus was compared to a homologous DNAvaccine candidate (Olsen et al., 1997). Collectively, these datademonstrate that a high degree of protection against influenza viruschallenge can be induced with recombinant HA or NA proteins, usingvarious experimental approaches and in different animal models.

Since previous research has shown that the surface influenzaglycoproteins, HA and NA, are the primary targets for elicitation ofprotective immunity against influenza virus and that M1 provides aconserved target for cellular immunity to influenza, a new vaccinecandidate may include these viral antigens as a protein macromolecularparticle, such as virus-like particles (VLPs). As vaccine products, VLPsoffer the advantage of being more immunogenic than subunit orrecombinant antigens and are able to stimulate both humoral and cellularimmune response (Grgacic and Anderson, 2006). Further, the particle withthese influenza antigens may display conformational epitopes that elicitneutralizing antibodies to multiple strains of influenza viruses.

Production of a non-infectious influenza virus strain for vaccinepurposes is one way to avoid inadvertent infection. Alternatively,virus-like particles (VLPs) as substitutes for the cultured virus havebeen investigated. VLPs mimic the structure of the viral capsid, butlack a genome, and thus cannot replicate or provide a means for asecondary infection.

Several studies have demonstrated that recombinant influenza proteinsself-assemble into VLPs in cell culture using mammalian expressionplasmids or baculovirus vectors (Gomez-Puertas et al., 1999; Neumann etal., 2000; Latham and Galarza, 2001). Gomez-Puertas et al. (1999)discloses that efficient formation of influenza VLP depends on theexpression levels of several viral proteins. Neumann et al. (2000)established a mammalian expression plasmid-based system for generatinginfectious influenza virus-like particles entirely from cloned cDNAs.Latham and Galarza (2001) reported the formation of influenza VLPs ininsect cells infected with recombinant baculovirus co-expressing HA, NA,M1, and M2 genes. These studies demonstrated that influenza virionproteins may self-assemble upon co-expression in eukaryotic cells.

Gomez-Puertas et al. (2000) teach that, in addition to the hemagglutinin(HA), the matrix protein (M1) of the influenza virus is essential forVLP budding from insect cells. However, Chen et al. (2007) teach that M1might not be required for VLP formation, and observed that efficientrelease of M1 and VLPs required the presence of HA and sialidaseactivity provided by NA. The NA cleaves the sialic acids of theglycoproteins at the surface of the cells producing the VLPs, andreleasing the VLPs in the medium.

Quan et al 2007 teaches that a VLP vaccine produced in a baculovirusexpression system (insect cell) induces a protective immunity againstsome strains of influenza virus (A/PR8/34 (H1N1)). The VLPs studied byQuan were observed to bud from the plasma membrane, and were consideredto be of the correct size and morphology, similar to those obtained in amammalian system (MDCK cells).

PCT Publications WO 2004/098530 and WO 2004/098533 teach expression ofNewcastle Disease Virus HN or Avian Influenza A/turkey/Wisconsin/68(H5N9) in transformed NT-1 (tobacco) cells in culture. Compositionscomprising the plant cell culture-expressed polypeptides elicit varyingimmune responses in rabbits and chickens.

Enveloped viruses may obtain their lipid envelope when ‘budding’ out ofthe infected cell and obtain the membrane from the plasma membrane, orfrom that of an internal organelle. Influenza virus particles and VLPsbud from the plasma membrane of the host cell. In mammalian orbaculovirus cell systems, for example, influenza buds from the plasmamembrane (Quan et al 2007). Only a few enveloped viruses are known toinfect plants (for example, members of the Topoviruses andRhabdoviruses). Of the known plant enveloped viruses, they arecharacterized by budding from internal membranes of the host cell, andnot from the plasma membrane. Although a small number of recombinantVLPs have been produced in plant hosts, none were derived from theplasma membrane, raising the question whether plasma membrane-derivedVLPs, including influenza VLPs can be produced in plants.

Current influenza VLP production technologies rely on the co-expressionof multiple viral proteins, and this dependence represents a drawback ofthese technologies since in case of a pandemic and of yearly epidemics,response time is crucial for vaccination. A simpler VLP productionsystem, for example, one that relies on the expression of only one or afew viral proteins without requiring expression of non-structural viralproteins is desirable to accelerate the development of vaccines.

In order to protect the world population from influenza and to stave offfuture pandemics, vaccine manufacturers will need to develop effective,rapid methods producing vaccine doses. The current use of fertilizedeggs to produce vaccines is insufficient and involves a lengthy process.

SUMMARY OF THE INVENTION

It is an object of the invention to provide improved influenza viruslike particles (VLPs).

According to the present invention there is provided a nucleic acidcomprising a nucleotide sequence encoding an antigen from an envelopedvirus operatively linked to a regulatory region active in a plant. Theantigen may be an influenza hemagglutinin (HA).

The HA may comprise a native, or a non-native signal peptide; thenon-native signal peptide may be a protein disulfide isomerase signalpeptide.

The HA encoded by the nucleic acid may be a type A influenza, a type Binfluenza, or is a subtype of type A influenza, selected from the groupcomprising H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and H16. In some aspects of the invention, the HA encoded by thenucleic acid may be from a type A influenza, and selected from the groupcomprising H1, H2, H3, H5, H6, H7 and H9.

The present invention also provides a method of producing influenzavirus like particles (VLPs) in a plant comprising:

-   -   a) introducing a nucleic acid encoding an antigen from an        enveloped virus, for example an influenza hemagglutinin (HA),        operatively linked to a regulatory region active in the plant,        into the plant, or portion thereof, and    -   b) incubating the plant or a portion therefore under conditions        that permit the expression of the nucleic acid, thereby        producing the VLPs.

The method may further comprise the steps of harvesting the plant andpurifying or separating the VLPs from the plant tissue.

The method may further comprise, in the step of introducing (step a), anucleic acid comprising a nucleotide sequence encoding one or more thanone chaperon protein.

The one or more than one chaperone proteins may be selected from thegroup comprising Hsp40 and Hsp70.

The present invention includes the above method wherein, in the step ofintroducing (step a), the nucleic acid may be either transientlyexpressed in the plant, or stably expressed in the plant. Furthermore,the VLPs may be purified using size exclusion chromatography.

According to another aspect of the present invention, there is provideda method of producing influenza virus like particles (VLPs) in a plantcomprising providing a plant, or a portion of a plant, comprising anucleic acid comprising a nucleotide sequence encoding an antigen froman enveloped virus operatively linked to a regulatory region active in aplant, and incubating the plant or portion of the plant under conditionsthat permit the expression of the nucleic acid, thereby producing theVLPs.

The method may further comprise the steps of harvesting the plant andpurifying or separating the VLPs from the plant tissue.

The present invention includes the above method, wherein following thestep of providing, a nucleic acid comprising a nucleotide sequenceencoding one or more than one chaperone protein operatively linked to aregulatory region active in a plant is introduced, and the plant orportion of the plant incubated under conditions that permit expressionof the nucleic acid, thereby producing the VLPs.

The one or more than one chaperone proteins may be selected from thegroup comprising Hsp40 and Hsp70.

The present invention includes the above method wherein, in the step ofintroducing (step a), the nucleic acid encoding the HA is stablyexpressed in the plant. Furthermore, the VLPs may be purified using sizeexclusion chromatography.

The present invention also provides a virus like particle (VLP)comprising an influenza virus HA protein and one or more than one lipidderived from a plant.

The HA protein of the VLP may be of a type A influenza, a type Binfluenza, or is a subtype of type A influenza HA selected from thegroup consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, and H16. In some aspects of the invention, the HA is froma type A influenza, selected from the group comprising H1, H2, H3, H5,H6, H7 and H9.

Also included in the present invention is a composition comprising aneffective dose of a VLP, the VLP comprising an influenza virus HAprotein, one or more than one plant lipid, and a pharmaceuticallyacceptable carrier.

The present invention also contemplates fragments or portions of HAproteins that form VLPs in a plant.

The present invention also pertains to a VLP comprising an influenzavirus HA bearing plant-specific N-glycans, or modified N-glycans. The HAprotein of the VLP may be of a type A influenza, a type B influenza, oris a subtype of type A influenza HA selected from the group consistingof H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, andH16. In some aspects of the invention, the HA is from a type Ainfluenza, selected from the group comprising H1, H2, H3, H5, H6, H7 andH9.

The VLP may comprise an HA protein of one, or more than one subtype,including H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15 or H16 or fragment or portion thereof. Examples of subtypescomprising such HA proteins include A/New Caledonia/20/99(H1N1)A/Indonesia/5/2006 (H5N1), A/chicken/New York/1995, A/herringgull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00,A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02,A/Turkey/Ontario/6118/68(H8N4), A/shoveler/Iran/G54/03,A/chicken/Germany/N/1949(H10N7), A/dull/England/56(H11N6),A/duck/Alberta/60/76(H12N5), A/Gull/Maryland/704/77(H13N6),A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H15N8), A/black-headedgull/Sweden/5/99(H16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34(H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1),A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),B/Malaysia/2506/2004, B/Florida/4/2006, A/Singapore/1/57 (H2N2),A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1),A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H7N7),A/HongKong/1073/99 (119N2)).

In an aspect of the invention, the HA protein may be an H1, H2, H3, H5,H6, H7 or H9 subtype. In an another aspect, the H1 protein may be fromthe A/New Caledonia/20/99 (H1N1), A/PuertoRico/8/34 (H1N1),A/Brisbane/59/2007 (H1N1), or A/Solomon Islands 3/2006 (H1N1) strain.The H3 protein may also be from the A/Brisbane 10/2007 (H3N2) orA/Wisconsin/67/2005 (H3N2) strain. In a further aspect of the invention,the H2 protein may be from the A/Singapore/1/57 (H2N2) strain. The H5protein may be from the A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004(H5N1), or A/Indonesia/5/2005 strain. In an aspect of the invention, theH6 protein may be from the A/Teal/HongKong/W312/97 (H6N1) strain. The H7protein may be from the A/Equine/Prague/56 (H7N7) strain. In an aspectof the invention, the H9 protein is from the A/HongKong/1073/99 (H9N2)strain. In a further aspect of the invention, the HA protein may be froman influenza virus may be a type B virus, including B/Malaysia/2506/2004or B/Florida/4/2006. Examples of amino acid sequences of the HA proteinsfrom H1, H2, H3, H5, H6, H7, H9 or B subtypes include SEQ ID NOs: 48-59.

The influenza virus HA protein may be H5 Indonesia.

The present invention also provides nucleic acid molecules comprisingsequences encoding an HA protein. The nucleic acid molecules may furthercomprise one or more regulatory regions operatively linked to thesequence encoding an HA protein. The nucleic acid molecules may comprisea sequence encoding an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, H16, B or C. In another aspect of the invention, theHA protein encoded by the nucleic acid molecule may be an H1, H2, H3,H5, H6, H7, H9, or B subtype. The H1 protein encoded by the nucleic acidmolecule is from the A/New Caledonia/20/99 (H1N1), A/PuertoRico/8/34(H1N1), A/Brisbane/59/2007 (H1N1), or A/Solomon Islands 3/2006 (H1N1)strain. In an aspect of the invention, the H3 protein encoded by thenucleic acid molecule may be from the A/Brisbane 10/2007 (H3N2), orA/Wisconsin/67/2005 (H3N2) strain. In a further aspect of the invention,the H2 protein encoded by the nucleic acid molecule may be from theA/Singapore/1/57 (H2N2) strain. The H5 protein encoded by the nucleicacid molecule may also be from the A/Anhui/1/2005 (H5N1),A/Vietnam/1194/2004 (H5N1), or A/Indonesia/5/2005 strain. In an aspectof the invention, the H6 protein encoded by the nucleic acid moleculemay be from the A/Teal/HongKong/W312/97 (H6N1) strain. The H7 proteinencoded by the nucleic acid molecule may also be from theA/Equine/Prague/56 (H7N7) strain. Additionally, the H9 protein encodedby the nucleic acid molecule may be from the A/HongKong/1073/99 (H9N2)strain. The HA protein from B subtype encoded by the nucleic acid may befrom the B/Florida/4/2006, or B/Malaysia/2506/2004 strain. Examples ofsequences of nucleic acid molecules encoding such HA proteins from H1,H2, H3, H5, H6, H7, H9 or B subtypes include SEQ ID NOs: 36-47 and60-73.

The nucleic acid sequence may encode the influenza virus HA protein H5Indonesia.

Regulatory regions that may be operatively linked to a sequence encodingan HA protein include those that are operative in a plant cell, aninsect cell or a yeast cell. Such regulatory regions may include aplastocyanin regulatory region, a regulatory region of Ribulose1,5-bisphosphate carboxylase/oxygenase (RuBisCO), chlorophyll a/bbinding protein (CAB), ST-LS1, a polyhedrin regulatory region, or a gp64regulatory region. Other regulatory regions include a 5′ UTR, 3′ UTR orterminator sequences. The plastocyanin regulatory region may be analfalfa plastocyanin regulatory region; the 5′ UTR, 3′UTR or terminatorsequences may also be alfalfa sequences.

A method of inducing immunity to an influenza virus infection in asubject, is also provided, the method comprising administering the viruslike particle comprising an influenza virus HA protein, one or more thanone plant lipid, and a pharmaceutically acceptable carrier. The viruslike particle may be administered to a subject orally, intradermally,intranasally, intramuscularly, intraperitoneally, intravenously, orsubcutaneously.

The present invention also pertains to a virus like particle (VLP)comprising one or more than one protein derived from a virus selectedfrom the group consisting of Influenza, Measles, Ebola, Marburg, andHIV, and one or more than one lipid derived from a non-sialylating hostproduction cell. The HIV protein may be p24, gp120 or gp41; theEbolavirus protein may be VP30 or VP35; the Marburg virus protein may beGp/SGP; the Measles virus protein may be H-protein or F-protein.

Additionally the present invention relates to a virus like particle(VLP) comprising an influenza virus HA protein and one or more than onehost lipid. For example if the host is insect, then the virus likeparticle (VLP) may comprise an influenza virus HA protein and one ormore than one insect lipid, or if the host is a yeast, then the viruslike particle (VLP) may comprise an influenza virus HA protein and oneor more than one yeast lipid.

The present invention also relates to compositions comprising VLPs oftwo or more strains or subtypes of influenza. The two or more subtypesor strains may be selected from the group comprising: A/NewCaledonia/20/99 (H1N1)A/Indonesia/5/2006 (H5N1), A/chicken/NewYork/1995, A/herring gull/DE/677/88 (H2N8), A/Texas/32/2003,A/mallard/MN/33/00, A/duck/Shanghai/1/2000, A/northernpintail/TX/828189/02, A/Turkey/Ontario/6118/68(H8N4),A/shoveler/Iran/G54/03, A/chicken/Germany/N/1949(H10N7),A/duck/England/56(H11N6), A/duck/Alberta/60/76(H12N5),A/Gull/Maryland/704/77(H13N6), A/Mallard/Gurjev/263/82,A/duck/Australia/341/83 (H15N8), A/black-headed gull/Sweden/5/99(H16N3),B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34 (H1N1),A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1), A/Brisbane10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004,B/Florida/4/2006, A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N1),A/Vietnam/1194/2004 (H5N1), A/Teal/HongKong/W312/97 (H6N1),A/Equine/Prague/56 (H7N7) or A/HongKong/1073/99 (H9N2)). The two or moresubtypes or strains of VLPs may be present in about equivalentquantities; alternately one or more of the subtypes or strains may bethe majority of the strains or subtypes represented.

The present invention pertains to a method for inducing immunity toinfluenza virus infection in an animal or target organism comprisingadministering an effective dose of a vaccine comprising one or more thanone VLP, the VLP produced using a non-sialyating host, for example aplant host, an insect host, or a yeast host. The vaccine may beadministered orally, intradermally, intranasally, intramusclarly,intraperitoneally, intravenously, or subcutaneously. The target organismmay be selected from the group comprising humans, primates, horses,pigs, birds (avian) water fowl, migratory birds, quail, duck, geese,poultry, chicken, camel, canine, dogs, feline, cats, tiger, leopard,civet, mink, stone marten, ferrets, house pets, livestock, mice, rats,seal, whales and the like.

The present invention provides a method for producing VLPs containinghemagglutinin (HA) from different influenza strains in a suitable hostcapable of producing a VLP, for example, a plant, insect, or yeast. VLPsthat are produced in plants contain lipids of plant origin, VLPsproduced in insect cells comprise lipids from the plasma membrane ofinsect cells (generally referred to as “insect lipids”), and VLPsproduced in yeast comprise lipids from the plasma membrane of yeastcells (generally referred to as “yeast lipids”).

The present invention also pertains to a plant, plant tissue or plantcell comprising a nucleic acid comprising a nucleotide sequence encodingan antigen from an enveloped virus operatively linked to a regulatoryregion active in a plant. The antigen may be an influenza hemagglutinin(HA).

The plant may further comprise a nucleic acid comprising a nucleotidesequence encoding one or more than one chaperone proteins operativelylinked to a regulatory region active in a plant. The one or more thanone chaperon proteins may be selected from the group comprising Hsp40and Hsp70.

The production of VLPs in plants presents several advantages over theproduction of these particles in insect cell culture. Plant lipids canstimulate specific immune cells and enhance the immune response induced.Plant membranes are made of lipids, phosphatidylcholine (PC) andphosphatidylethanolamine (PE), and also contain glycosphingolipids thatare unique to plants and some bacteria and protozoa. Sphingolipids areunusual in that they are not esters of glycerol like PC or PE but ratherconsist of a long chain amino alcohol that forms an amide linkage to afatty acid chain containing more than 18 carbons. PC and PE as well asglycosphingolipids can bind to CD1 molecules expressed by mammalianimmune cells such as antigen-presenting cells (APCs) like dentriticcells and macrophages and other cells including B and T lymphocytes inthe thymus and liver (Tsuji M., 2006). Furthermore, in addition to thepotential adjuvant effect of the presence of plant lipids, the abilityof plant N-glycans to facilitate the capture of glycoprotein antigens byantigen presenting cells (Saint-Jore-Dupas, 2007), may be advantageousof the production of VLPs in plants.

Without wishing to be bound by theory, it is anticipated that plant-madeVLPs will induce a stronger immune reaction than VLPs made in othermanufacturing systems and that the immune reaction induced by theseplant-made VLPs will be stronger when compared to the immune reactioninduced by live or attenuated whole virus vaccines.

Contrary to vaccines made of whole viruses, VLPs provide the advantageas they are non-infectious, thus restrictive biological containment isnot as significant an issue as it would be working with a whole,infectious virus, and is not required for production. Plant-made VLPsprovide a further advantage again by allowing the expression system tobe grown in a greenhouse or field, thus being significantly moreeconomical and suitable for scale-up.

Additionally, plants do not comprise the enzymes involved insynthesizing and adding sialic acid residues to proteins. VLPs may beproduced in the absence of neuraminidase (NA), and there is no need toco-express NA, or to treat the producing cells or extract with sialidase(neuraminidase), to ensure VLP production in plants.

The VLPs produced in accordance with the present invention do notcomprise M1 protein which is known to bind RNA. RNA is a contaminant ofthe VLP preparation and is undesired when obtaining regulatory approvalfor the VLP product.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1A shows a sequence of an alfalfa plastocyanin-based expressioncassette used for the expression of H1 from strain A/New Caledonia/20/99(H1N1) in accordance with an embodiment of the present invention (SEQ IDNO:8). Protein disulfide isomerase (PDI) signal peptide is underlined.BglII (AGATCT) and SacI (GAGCTC) restriction sites used for cloning areshown in bold. FIG. 1B shows a schematic diagram of functional domainsof influenza hemagglutinin. After cleavage of HA0, HA1 and HA2 fragmentsremain bound together by a disulfide bridge.

FIG. 2A shows a representation of plasmid 540 assembled for theexpression of HA subtype H1 from strain A/New Caledonia/20/99 (H1N1).FIG. 2B shows a representation of plasmid 660 assembled for theexpression of HA subtype H5 from strain A/Indonesia/5/2005 (H5N1).

FIG. 3 shows a size exclusion chromatography of protein extracts fromleaves producing hemagglutinin H1 or H5. FIG. 3A show the elutionprofile of Blue Dextran 2000 (triangles) and proteins (diamonds). FIG.3B shows immunodetection (western blot; anti H1) of H1 (A/NewCaledonia/20/99 (H1N1)) elution fractions following size exclusionchromatography (S500HR beads). FIG. 3C show the elution profile of H5;Blue Dextran 2000 (triangles) and proteins (diamonds). FIG. 3D showsimmunodetection (western blot; anti H5) of H5 (A/Indonesia/5/2005(H5N1)) elution fractions following size exclusion chromatography(S500HR beads).

FIG. 4A shows the sequence encoding the N terminal fragment of H1 (A/NewCaledonia/20/99 (H1N1)) (SEQ ID NO:1). FIG. 4B shows the sequenceencoding the C terminal fragment of H1 (A/New Caledonia/20/99 (H1N1))(SEQ ID NO:2).

FIG. 5 shows the complete sequence encoding HA0 of H1 (A/NewCaledonia/20/99 (H1N1)) (SEQ ID NO:28).

FIG. 6 shows the sequence encoding H5 (A/Indonesia/5/2005 (H5N1))flanked by a HindIII site immediately upstream of the initial ATG, and aSacI site immediately downstream of the stop (TAA) codon (SEQ ID NO:3).

FIG. 7A shows the sequence of the primer Plasto-443c (SEQ ID NO:4). FIG.7B shows the sequence of primer SpHA(Ind)-Plasto.r (SEQ ID NO:5). FIG.7C shows the sequence of primer Plasto-SpHA(Ind).c (SEQ ID NO:6). FIG.7D shows the sequence of primer HA(Ind)-Sac.r (SEQ ID NO:7).

FIG. 8A shows the amino acid sequence of the H1 (A/New Caledonia/20/99(H1N1)) peptide sequence (SEQ ID NO:9). FIG. 8B shows the amino acidsequence of H15 (A/Indonesia/5/2005 (H5N1)) peptide sequence (SEQ IDNO:10). Native signal peptide is indicated in bold.

FIG. 9 shows the nucleotide sequence of HA of influenza A subtype H7(SEQ ID No: 11).

FIG. 10A shows the nucleotide sequence of Influenza A HA, subtype H2(SEQ ID NO:12). FIG. 10B shows the nucleotide sequence of Influenza A HAsubtype H3 (SEQ ID NO:13). FIG. 10C shows the nucleotide sequence ofInfluenza A HA subtype H4 (SEQ ID NO:14). FIG. 10D shows the nucleotidesequence of Influenza A HA subtype H5 (SEQ ID NO:15). FIG. 10E shows thenucleotide sequence of Influenza A HA subtype H6 (SEQ ID NO:16). FIG.10F shows the nucleotide sequence of Influenza A HA subtype H8 (SEQ IDNO:17). FIG. 10G shows the nucleotide sequence of Influenza A HA subtypeH9 (SEQ ID NO:18). FIG. 10H shows the nucleotide sequence of Influenza AHA subtype H10 (SEQ ID NO:19). FIG. 10I shows the nucleotide sequence ofInfluenza A HA subtype H11 (SEQ ID NO:20). FIG. 10J shows the nucleotidesequence of Influenza A HA subtype H12 (SEQ ID NO:21). FIG. 10K showsthe nucleotide sequence of Influenza A HA subtype H13 (SEQ ID NO:22).FIG. 10L shows the nucleotide sequence of Influenza A HA subtype H14(SEQ ID NO:23). FIG. 10M shows the nucleotide sequence of Influenza A HAsubtype H15 (SEQ ID NO:24). FIG. 10N shows the nucleotide sequence ofInfluenza A HA subtype H16 (SEQ ID NO:25). FIG. 10O shows the nucleotidesequence of Influenza B HA (SEQ ID NO:26). FIG. 10P shows the nucleotidesequence of Influenza C HA (SEQ ID NO:27). FIG. 10Q shows the nucleotidesequence of primer XmaI-pPlas.c (SEQ ID NO: 29). FIG. 10R shows thenucleotide sequence of primer SacI-ATG-pPlas.r (SEQ ID NO: 30). FIG. 10Sshows the nucleotide sequence of primer SacI-PlasTer.c (SEQ ID NO: 31).FIG. 10T shows the nucleotide sequence of primer EcoRI-PlasTer.r (SEQ IDNO: 32).

FIG. 11 shows a schematic representation of several constructs as usedherein. Construct 660 comprises the nucleotide sequence to encode the HAsubtype H5 (A/Indonesia/5/2005 (H5N1)) under operatively linked to theplastocyanin promoter (plasto) and terminator (Pter); construct 540comprises the nucleotide sequence to encode the HA subtype H1 (A/NewCaledonia/20/99 (H1N1)) in combination with an alfalfa protein disulfideisomerase signal peptide (SP PDI), and is operatively linked to aplastocyanin promoter (Plasto) and terminator (Pter); construct 544assembled for the expression of HA subtype H1 (A/New Caledonia/20/99(H1N1)), the nucleotide sequence encoding H1 is combined with an alfalfaprotein disulfide isomerase signal peptide (SP PDI) and an GCN4pIIleucine zipper (in place of the transmembrane domain and cytoplasmictail of HI) and operatively linked to the plastocyanin promoter (Plasto)and terminator (Pter); and construct 750 for the expression of M1 codingregion from influenza A/PR/8/34 is combined to the tobacco etch virus(TEV) 5′UTR, and operatively linked with the double 35S promoter and Nosterminator.

FIG. 12 shows immunodetection of H5 (A/Indonesia/5/2005 (H5N1)), usinganti-H5 (Vietnam) antibodies, in protein extracts from N. benthamianaleaves transformed with construct 660 (lane 3). Commercial H5 frominfluenza A/Vietnam/1203/2004 was used as positive control of detection(lane 1), and a protein extract from leaves transformed with an emptyvector were used as negative control (lane 2).

FIG. 13 shows characterization of hemagglutinin structures by sizeexclusion chromatography. Protein extract from separate biomassesproducing H5 (A/Indonesia/5/2005 (H5N1)), H1 (A/New Caledonia/20/99(H1N1)), soluble H1, or H1 and M1 were separated by gel filtration onS-500 HR. Commercial H1 (A/New Caledonia/20/99 (H1N1)) in the form ofrosettes was also fractionated (H1 rosette). FIG. 13A shows elutionfractions analyzed for relative protein content (Relative ProteinLevel—a standard protein elution profile of a biomass fractionation isshown). Blue Dextran 2000 (2 MDa reference standard) elution peak isindicated. FIG. 13B shows elution fractions analyzed for the presence ofhemagglutinin by immunoblotting with anti-H5 (Vietnam) antibodies (forH5). FIG. 13C shows elution fractions analyzed for anti-influenza Aantibodies for H1. FIG. 13D shows elution fractions analyzed foranti-influenza A antibodies for soluble H1. FIG. 13E shows elutionfractions analyzed for anti-influenza A antibodies for H1 rosette. FIG.13F shows elution fractions analyzed for anti-influenza A antibodies forH1+M1.

FIG. 14 shows concentration of influenza H5 (A/Indonesia/5/2005 (H5N1))structures by sucrose gradient centrifugation and electron microscopyexamination of hemagglutinin-concentrated fractions. FIG. 14A showscharacterization of fractions from sucrose density gradientcentrifugation. Each fraction was analyzed for the presence of H5 byimmunoblotting using anti-H5 (Vietnam) antibodies (upper panel), and fortheir relative protein content and hemagglutination capacity (graph).FIG. 14B shows negative staining transmission electron microscopyexamination of pooled fractions 17, 18 and 19 from sucrose gradientcentrifugation. The bar represents 100 nm.

FIG. 15 shows purification of influenza H5 VLPs. FIG. 15A showsCoomassie Blue stained SDS-PAGE analysis of protein content in theclarification steps—lane 1, crude extract; lane 2, pH 6-adjustedextract; lane 3, heat-treated extract; lane 4, DE-filtrated extract; thefetuin affinity purification steps: lane 5, load; lane 6, flowthrough;lane 7, elution (10× concentrated). FIG. 15B shows negative stainingtransmission electron microscopy examination of the purified H5 VLPsample. The bar represents 100 nm. FIG. 15C shows isolated H5 VLPenlarged to show details of the structure. FIG. 15D shows the H5 VLPproduct on a Coomassie-stained reducing SDS-PAGE (lane A) and Westernblot (lane B) using rabbit polyclonal antibody raised against HA fromstrain A/Vietnam/1203/2004 (H5N1).

FIG. 16 shows a nucleotide sequence for Influenza A virus (A/NewCaledonia/20/99(H1N1)) hemagglutinin (HA) gene, complete cds. GenBankAccession No. AY289929 (SEQ ID NO: 33).

FIG. 17 shows a nucleotide sequence for Medicago sativa mRNA for proteindisulfide isomerase. GenBank Accession No. Z11499 (SEQ ID NO: 34).

FIG. 18 shows a nucleotide sequence for Influenza A virus (A/PuertoRico/8/34(H1N1)) segment 7, complete sequence. GenBank Accession No.NC_002016.1 (SEQ ID NO: 35).

FIG. 19 shows localization of VLP accumulation by positive stainingtransmission electron microscopy observation of H5 producing tissue. CW:cell wall, ch: chloroplast, pm: plasma membrane, VLP: virus-likeparticle. The bar represents 100 nm.

FIG. 20 shows induction of serum antibody responses 14 days after boostin Balb/c mice vaccinated with plant-made influenza 115 VLP(A/Indonesia/5/2005 (H5N1)) or recombinant soluble H5(A/Indonesia/5/2005 (H5N1)). FIG. 20(A) Antibody responses of miceimmunized through intramuscular injection. FIG. 20(B) Antibody responsesof mice immunized through intranasal administration. Antibody responseswere measured against inactivated whole H5N1 viruses (A/Indonesia/5/05).GMT: geometric mean titer. Values are the GMT (In) of reciprocalend-point titers of five mice per group. Bars represent meandeviation. * p<0.05 compared to recombinant soluble H5.

FIG. 21 shows hemagglutination inhibition antibody response (HAI) 14days after boost in Balb/c mice vaccinated with plant-made influenza H5VLP (A/Indonesia/5/2005 (H5N1)) or recombinant soluble H5(A/Indonesia/5/2005 (H5N1)). FIG. 21(A) Antibody responses of miceimmunized through intramuscular injection. FIG. 21(B) Antibody responsesof mice immunized through intranasal administration. HAI antibodyresponses were measured using inactivated whole H5N1 viruses(A/Indonesia/5/05). GMT: geometric mean titer. Values are the GMT (ln)of reciprocal end-point titers of five mice per group. Bars representmean deviation. * p<0.05 and ** p<0.01 compared to recombinant solubleH5.

FIG. 22 shows the effect of adjuvant on immunogenicity of the VLPs inBalb/c mice. FIG. 22(A) Effect of alum on mice immunized throughintramuscular injection. FIG. 22(B) Effect of Chitosan on mice immunizedthrough intranasal administration. HAI antibody responses were measuredusing inactivated whole H5N1 viruses (A/Indonesia/5/05). GMT: geometricmean titer. Values are the GMT (ln) of reciprocal end-point titers offive mice per group. Bars represent mean deviation. * p<0.05 compared tothe corresponding recombinant soluble H5.

FIG. 23 shows antibody response to H5 VLP (A/Indonesia/5/2005 (H5N1))administration. FIG. 23(A) Anti-Indonesia/5/05 immunoglobulin isotype inmice immunized through intramuscular administration, 30 days afterboost. Values are the GMT (log₂) of reciprocal end-point titers of fivemice per group. ELISA performed using whole inactivated H5N1(A/Indonesia/5/2005) viruses as the coating agent. Bars represent meandeviation. * p<0.05, ** p<0.001 compared to the correspondingrecombinant soluble H5 (A/Indonesia/5/2005 (H5N1)). FIG. 23(B) Antibodytiters against whole inactivated viruses (A/Indonesia/5/2005 (H5N1) and(A/Vietnam/1194/04 (H5N1))). All groups are statistically different tonegative control.

FIG. 24 shows antibody titer against homologous whole inactivatedviruses (A/Indonesia/5/05), 14 days weeks after first dose (week 2), 14days after boost (week 5) or 30 days after boost (week 7) from Balb/cmice immunized with H15 VLP (A/Indonesia/5/2005 (H5N1)). GMT: geometricmean titer. Values are the GMT (ln) of reciprocal end-point titers offive mice per group. * p<0.05 compared to recombinant soluble H5.

FIG. 25 shows in vitro cross-reactivity of serum antibodies from Balb/cmice immunized with H5 VLP (A/Indonesia/5/2005 (H5N1)) 30 days afterboost. (A) Antibody titers whole inactivated viruses. (B)Hemagglutination-inhibition titers against various whole inactivatedviruses. Values are the GMT (ln) of reciprocal end-point titers of fivemice per group. Bars represent mean deviation. All groups arestatistically different to negative control. * p<0.05 compared to thecorresponding recombinant soluble H5. All values less than 10 were givenan arbitrary value of 5 (1.6 for ln) and are considered negative.

FIG. 26 shows efficacy of the plant made H5 VLP (A/Indonesia/5/2005(H5N1)). (A) Survival rate of mice after challenge with 1000 LD₅₀(4.09×10⁶ CCID₅₀) of the influenza strain A/Turkey/582/06 (H5N1) (B)Body weight of immunised mice after challenge. Values are the mean bodyweight of surviving mice.

FIG. 27 shows origin of plant-derived influenza VLPs. (A) Polar lipidcomposition of purified influenza VLPs. Lipids contained in anequivalent of 40 μg of proteins, were extracted from VLP as described,separated by HP-TLC, and compared to the migration profile of lipidsisolated from highly purified tobacco plasma membrane (PM). Lipidabbreviations are as following: DGDG, Digalactosyldiacylglycerol;gluCER, glucosyl-ceramide; PA, phosphatic acid; PC, phosphatidylcholine;PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI,phosphatidylinositol; PS, phosphatidylserine; SG, Steryl-glycoside. (B)Neutral lipid composition of purified influenza VLPs. Lipids containedin an equivalent of 20 μg of proteins were extracted from VLP asdescribed, separated by HP-TLC and compared to the migration ofsitosterol. (C) Immunodetection of the plasma membrane marker protonpump ATPase (PMA) in purified VLPs and highly-purified PM from tobaccoleaves (PM_(L)) and BY2 tobacco cells (PM_(BY2)). Eighteen micrograms ofprotein were loaded in each lane.

FIG. 28 shows the sequence spanning from DraIII to SacI sites of clone774-nucleotide sequence of A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 36).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 29 shows the sequence spanning from DraIII to SacI sites of clone775-nucleotide sequence of A/Solomon Islands 3/2006 (H1N1) (SEQ ID NO:37). The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 30 shows the sequence spanning from DraIII to SacI sites of clone776-nucleotide sequence of A/Brisbane 10/2007 (H3N2) (SEQ ID NO: 38).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 31 shows the sequence spanning from DraIII to SacI sites of clone777-nucleotide sequence of A/Wisconsin/67/2005 (H3N2) (SEQ ID NO: 39).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 32 shows the sequence spanning from DraIII to SacI sites of clone778-nucleotide sequence of B/Malaysia/2506/2004 (SEQ ID NO: 40). Thecoding sequence is flanked by a plastocyanin regulatory region, startingwith a DraIII restriction site at the 5′ end and by a stop codon and aSacI site at the 3′ end. Restriction sites are underlined; ATG is inbold and underlined.

FIG. 33 shows the sequence spanning from DraIII to SacI sites of clone779-nucleotide sequence of B/Florida/4/2006 (SEQ ID NO: 41). The codingsequence is flanked by a plastocyanin regulatory region, starting with aDraIII restriction site at the 5′ end and by a stop codon and a SacIsite at the 3′ end. Restriction sites are underlined; ATG is in bold andunderlined.

FIG. 34 shows the sequence spanning from DraIII to SacI sites of clone780-nucleotide sequence of A/Singapore/1/57 (H2N2) (SEQ ID NO: 42). Thecoding sequence is flanked by a plastocyanin regulatory region, startingwith a DraIII restriction site at the 5′ end and by a stop codon and aSacI site at the 3′ end. Restriction sites are underlined; ATG is inbold and underlined.

FIG. 35 shows the sequence spanning from DraIII to SacI sites of clone781-nucleotide sequence of A/Anhui/1/2005 (H5N1) (SEQ ID NO: 43). Thecoding sequence is flanked by a plastocyanin regulatory region, startingwith a DraIII restriction site at the 5′ end and by a stop codon and aSacI site at the 3′ end. Restriction sites are underlined; ATG is inbold and underlined.

FIG. 36 shows the sequence spanning from DraIII to SacI sites of clone782-nucleotide sequence of A/Vietnam/1194/2004 (H5N1) (SEQ ID NO: 44).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 37 shows the sequence spanning from DraIII to SacI sites of clone783-nucleotide sequence of A/Teal/HongKong/W312/97 (H6N1) (SEQ ID NO:45). The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 38 shows the sequence spanning from DraIII to SacI sites of clone784-nucleotide sequence of A/Equine/Prague/56 (H7N7) (SEQ ID NO: 46).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 39 shows the sequence spanning from DraIII to SacI sites of clone785-nucleotide sequence of A/HongKong/1073/99 (H9N2) (SEQ ID NO: 47).The coding sequence is flanked by a plastocyanin regulatory region,starting with a DraIII restriction site at the 5′ end and by a stopcodon and a SacI site at the 3′ end. Restriction sites are underlined;ATG is in bold and underlined.

FIG. 40A shows the amino acid sequence (SEQ ID NO: 48) of thepolypeptide translated from clone 774 (A/Brisbane/59/2007 (H1N1)). Theopen reading frame of clone 774 starts with the ATG indicated in FIG.28. FIG. 40B shows the amino acid sequence (SEQ ID NO: 49) of thepolypeptide translated from clone 775 (A/Solomon Islands 3/2006 (H1N1)).The open reading frame of clone 775 starts with the ATG indicated inFIG. 29.

FIG. 41A shows the amino acid sequence (SEQ ID NO: 50) of thepolypeptide translated from clone 776 (A/Brisbane/10/2007 (H3N2)). Theopen reading frame of clone 776 starts with the ATG indicated in FIG.30. FIG. 41B shows the amino acid sequence (SEQ ID NO: 51) of thepolypeptide translated from clone 777 (A/Wisconsin/67/2005 (H3N2)). Theopen reading frame of clone 777 starts with the ATG indicated in FIG.31.

FIG. 42A shows the amino acid sequence (SEQ ID NO: 52) of thepolypeptide translated from clone 778 (B/Malaysia/2506/2004). The openreading frame of clone 778 starts with the ATG indicated in FIG. 32.FIG. 42B shows the amino acid sequence (SEQ ID NO: 53) of thepolypeptide translated from clone 779 (B/Florida/4/2006). The openreading frame of clone 779 starts with the ATG indicated in FIG. 33.

FIG. 43A shows the amino acid sequence (SEQ ID NO: 54) of thepolypeptide translated from clone 780 (A/Singapore/1/57 (H2N2)). Theopen reading frame of clone 780 starts with the ATG indicated in FIG.34. FIG. 43B shows the amino acid sequence (SEQ ID NO: 55) of thepolypeptide translated from clone 781 (A/Anhui/1/2005 (115N1)). The openreading frame of clone 781 starts with the ATG indicated in FIG. 35.

FIG. 44A shows the amino acid sequence (SEQ ID NO: 56) of thepolypeptide translated from clone 782 (A/Vietnam/1194/2004 (H5N1)). Theopen reading frame of clone 782 starts with the ATG indicated in FIG.36. FIG. 44B shows the amino acid sequence (SEQ ID NO: 57) of thepolypeptide translated from clone 783 (A/Teal/HongKong/W312/97 (H6N1)).The open reading frame of clone 783 starts with the ATG indicated inFIG. 37.

FIG. 45A shows the amino acid sequence (SEQ ID NO: 58) of thepolypeptide translated from clone 784 (A/Equine/Prague/56 (H7N7)). Theopen reading frame of clone 784 starts with the ATG indicated in FIG.38. FIG. 45B shows the amino acid sequence (SEQ ID NO: 59) of thepolypeptide translated from clone 785 (A/HongKong/1073/99 (H9N2)). Theopen reading frame of clone 785 starts with the ATG indicated in FIG.39.

FIG. 46 shows immunodetection (western blot) of elution fractions 7-17of plant-produced VLPs, following size exclusion chromatography. Theelution peak (fraction 10) of BlueDextran is indicated by the arrow.Hemagglutinin subtypes H1, H2, H3, H5, H6 and H9 are shown.Hemagglutinin is detected in fractions 7-14, corresponding to theelution of VLPs.

FIG. 47 shows an immunoblot analysis of expression of a series ofhemagglutinin from annual epidemic strains. Ten and twenty micrograms ofleaf protein extracts were loaded in lanes 1 and 2, respectively, forplants expressing HA from various influenza strains (indicated at thetop of the immunoblots).

FIG. 48A shows an immunoblot analysis of expression of a series of H5hemagglutinins from potential pandemic strains. Ten and twentymicrograms of protein extracts were loaded in lanes 1 and 2,respectively. FIG. 48B shows an immunoblot analysis of expression of H2,H7 and H9 hemagglutinin from selected influenza strains. Ten and twentymicrograms of protein extracts were loaded in lanes 1 and 2,respectively.

FIG. 49 shows an immunoblot of H5 from strain A/Indonesia/5/2005 inprotein extracts from Nicotiana tabacum leaves, agroinfiltrated withAGL1/660. Two plants (plant 1 and plant 2) were infiltrated and 10 and20 μg of soluble protein extracted from each plant were loaded in lanes1 and 2, respectively.

FIG. 50 shows the in vitro cross-reactivity of serum antibodies.Hemagglutination-inhibition (HI) titers in ferret sera, 14 days (A)after 1^(st) immunization and (B) after 2nd boost with plant-madeinfluenza H5 VLP (A/Indonesia/5/2005 (H5N1)). HAI antibody responseswere measured using the following inactivated whole H5N1 viruses:A/turkey/Turkey/1/05, A/Vietnam/1194/04, A/Anhui/5/05 and the homologousstrain A/Indonesia/5/05. Values are the GMT (log₂) of reciprocalend-point titers of five ferrets per group. Diagonalstripe—A/Indonesia/6/06 (clade 2.1.3); checked—A/turkey/Turkey/1/05(clade 2.2); white bar—A/Vietnam/1194/04 (clade 1); black barA/Anhui/5/05. Responders are indicated. Bars represent mean deviation.

FIG. 51 shows the nucleic acid sequence (SEQ ID NO: 60) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 from A/Indonesia/5/2005 (Construct#660), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 52 shows the nucleic acid sequence (SEQ ID NO: 61) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 from A/New Caledonia/20/1999(Construct #540), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 53 shows the nucleic acid sequence (SEQ ID NO: 62) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 from A/Brisbane/59/2007 (construct#774), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 54 shows the nucleic acid sequence (SEQ ID NO: 63) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 from A/Solomon Islands/3/2006 (H1N1)(construct #775), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 55 shows the nucleic acid sequence (SEQ ID NO: 64) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H2 from A/Singapore/1/57 (H2N2)(construct #780), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 56 shows the nucleic acid sequence (SEQ ID NO: 65) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 from A/Anhui/1/2005 (H5N1)(Construct #781), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 57 shows the nucleic acid sequence (SEQ ID NO: 66) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 from A/Vietnam/1194/2004 (H5N1)(Construct #782), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 58 shows the nucleic acid sequence (SEQ ID NO: 67) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H6 from A/Teal/Hong Kong/W312/97 (H6N1)(Construct #783), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 59 shows the nucleic acid sequence (SEQ ID NO: 68) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H9 from A/Hong Kong/1073/99 (H9N2)(Construct #785), alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 60 shows the nucleic acid sequence (SEQ ID NO: 69) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H3 from A/Brisbane/10/2007 (H3N2),alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 61 shows the nucleic acid sequence (SEQ ID NO: 70) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H3 from A/Wisconsin/67/2005 (H3N2),alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 62 shows the nucleic acid sequence (SEQ ID NO: 71) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H7 from A/Equine/Prague/56 (H7N7),alfalfa plastocyanin 3′ UTR and terminator sequences.

FIG. 63 shows the nucleic acid sequence (SEQ ID NO: 72) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of HA from B/Malaysia/2506/2004, alfalfaplastocyanin 3′ UTR and terminator sequences.

FIG. 64 shows the nucleic acid sequence (SEQ ID NO: 73) of an HAexpression cassette comprising alfalfa plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of HA from B/Florida/4/2006, alfalfaplastocyanin 3′ UTR and terminator sequences.

FIG. 65 shows a consensus amino acid sequence (SEQ ID NO: 74) for HA ofA/New Caledonia/20/99 (H1N1) (encoded by SEQ ID NO: 33),A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 48), A/Solomon Islands/3/2006(H1N1) (SEQ ID NO: 49) and SEQ ID NO: 9. X1 (position 3) is A or V; X2(position 52) is D or N; X3 (position 90) is K or R; X4 (position 99) isK or T; X5 (position 111) is Y or H; X6 (position 145) is V or T; X7(position 154) is E or K; X8 (position 161) is R or K; X9 (position 181)is V or A; X10 (position 203) is D or N; X11 (position 205) is R or K;X12 (position 210) is T or K; X13 (position 225) is R or K; X14(position 268) is W or R; X15 (position 283) is T or N; X16 (position290) is E or K; X17 (position 432) is I or L; X18 (position 489) is N orD.

FIG. 66 shows amino acid sequence (SEQ ID NO: 75) of H1 New Caledonia(AAP34324.1) encoded by SEQ ID NO: 33.

FIG. 67 shows the amino acid sequence (SEQ ID NO: 76) of H1 Puerto Rico(NC_0409878.1) encoded by SEQ ID NO: 35

FIG. 68 shows the nucleic acid sequence of a portion of expressioncassette number 828, from PacI (upstream promoter) to AscI (immediatelydownstream NOS terminator). CPMV HT 5′UTR sequence underlined withmutated ATG. ApaI restriction site (immediately upstream of ATG ofprotein coding sequence to be express, in this case C5-1 kappa lightchain.)

FIG. 69 shows the nucleic acid sequence of a portion of construct number663, from HindIII (in the multiple cloning site, upstream of theplastocyanin promoter) to EcoRI (immediately downstream of theplastocyanin terminator). H5 (from A/Indonesia/5/2005) coding sequencein fusion with PDI SP is underlined.

FIG. 70 shows the nucleic acid sequence of a portion of construct number787, from HindIII (in the multiple cloning site, upstream of theplastocyanin promoter) to EcoRI (immediately downstream of theplastocyanin terminator). H1 (from A/Brisbane/59/2007) coding sequencein fusion with PDI SP is underlined.

FIG. 71 shows the nucleic acid sequence of a portion of construct number790, from HindIII (in the multiple cloning site, upstream of theplastocyanin promoter) to EcoRI (immediately downstream of theplastocyanin terminator). H3 (from A/Brisbane/10/2007) coding sequencein fusion with PDI SP is underlined.

FIG. 72 shows the nucleic acid sequence of a portion of construct number798, from HindIII (in the multiple cloning site, upstream of theplastocyanin promoter) to EcoRI (immediately downstream of theplastocyanin terminator). HA from B/Florida/4/2006 coding sequence infusion with PDI SP is underlined.

FIG. 73 shows the nucleic acid sequence of a portion of construct number580, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H1 (from A/NewCaledonia/20/1999) in fusion with PDI SP is underlined.

FIG. 74 shows the nucleic acid sequence of a portion of construct number685, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H5 fromA/Indonesia/5/2005 is underlined.

FIG. 75 shows the nucleic acid sequence of a portion of construct number686, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H5 fromA/Indonesia/5/2005 in fusion with PDI SP is underlined.

FIG. 76 shows the nucleic acid sequence of a portion of construct number732, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H1 fromA/Brisbane/59/2007 is underlined.

FIG. 77 shows the nucleic acid sequence of a portion of construct number733, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H1 fromA/Brisbane/59/2007 in fusion with PDI SP is underlined.

FIG. 78 shows the nucleic acid sequence of a portion of construct number735, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H3 fromA/Brisbane/10/2007 is underlined.

FIG. 79 shows the nucleic acid sequence of a portion of construct number736, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of H3 fromA/Brisbane/10/2007 in fusion with PDI SP is underlined.

FIG. 80 shows the nucleic acid sequence of a portion of construct number738, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of HA fromB/Florida/4/2006 is underlined.

FIG. 81 shows the nucleic acid sequence of a portion of construct number739, from PacI (upstream of the 35S promoter) to AscI (immediatelydownstream of the NOS terminator). Coding sequence of HA fromB/Florida/4/2006 in fusion with PDI SP is underlined.

FIG. 82 shows a nucleic acid sequence encoding Msj1 (SEQ ID NO: 114).

FIG. 83 shows the nucleic acid sequence of a portion of construct numberR850, from HindIII (in the multiple cloning site, upstream of thepromoter) to EcoRI (immediately downstream of the NOS terminator). HSP40coding sequence is underlined.

FIG. 84 shows the nucleic acid sequence of a portion of construct numberR860, from HindIII (in the multiple cloning site, upstream of thepromoter) to EcoRI (immediately downstream of the NOS terminator). HSP70coding sequence is underlined.

FIG. 85 shows the nucleic acid sequence of a portion of construct numberR870, from HindIII (in the multiple cloning site, upstream of thepromoter) to EcoRI (immediately downstream of the NOS terminator). HSP40coding sequence is in underlined italic and HSP70 coding sequence isunderlined. A) nucleotides 1-5003; B) nucleotides 5004-9493.

FIG. 86 shows a schematic representation of construct R472.

FIG. 87 shows an immunoblot analysis of expression of HA using a signalpeptide from alfalfa protein disulfide isomerase. Twenty micrograms ofleaf protein extract obtained from 3 separate plants were loaded on theSDS-PAGE except for the H1 (A/New Caledonia/20/99 (H1N1)) where fivemicrograms were used. The indicated controls (whole inactivated virus(WIV) of homologous strain) were spiked in five or twenty micrograms ofmock-infiltrated plants. a) Expression of H1 from A/NewCaledonia/20/99), b) expression of H1 from A/Brisbane/59/2007, c)expression of H3 from A/Brisbane/10/2007, d) expression of H5 fromA/Indonesia/5/2005, e) expression of HA from B/Florida/4/2006. Thearrows indicate the immunoband corresponding to HA0. SP WT: nativesignal peptide, PS PDI: alfalfa PDI signal peptide.

FIG. 88 shows a comparison of HA expression strategies by immunoblotanalysis of leaf protein extracts. HA was produced using plastocyanin-or CPMV-HT-based cassettes. For CPMV-HT, the wild-type HA signal peptideand the signal peptide from alfalfa PDI were also compared. Twentymicrograms of protein extract were loaded on the SDS-PAGE for HA subtypeanalyzed except for the H1 New Caledonia for which five micrograms ofproteins were loaded. a) Expression of H1 from A/New Caledonia/20/1999,b) expression of H1 from A/Brisbane/59/2007, c) expression of H3 fromA/Brisbane/10/2007, d) expression of H5 from A/Indonesia/5/2005, and e)expression of B from B/Florida/4/2006. The arrows indicate theimmunoband corresponding to HA0; specific Agrobacterium strainscomprising the specific vectors used for HA expression are indicated atthe top of the lanes.

FIG. 89 shows an immunoblot of HA accumulation when co-expressed withHsp 40 and Hsp70. H1 New Caledonia (AGL1/540) and H3 Brisbane (AGL1/790)were expressed alone or co-expressed with AGL1/R870. HA accumulationlevel was evaluated by immunoblot analysis of protein extracts frominfiltrated leaves. Whole inactivated virus (WIV) of strain A/NewCaledonia/20/99 or Brisbane/10/2007 were used as controls.

DETAILED DESCRIPTION

The present invention relates to the production of virus-like particles.More specifically, the present invention is directed to the productionof virus-like particles comprising influenza antigens.

The following description is of a preferred embodiment.

The present invention provides a nucleic acid comprising a nucleotidesequence encoding an antigen from an enveloped virus, for example, theinfluenza hemagglutinin (HA), operatively linked to a regulatory regionactive in a plant.

Furthermore, the present invention provides a method of producing viruslike particles (VLPs) in a plant. The method involves introducing anucleic acid encoding an antigen operatively linked to a regulatoryregion active in the plant, into the plant, or portion of the plant, andincubating the plant or a portion of the plant under conditions thatpermit the expression of the nucleic acid, thereby producing the VLPs.

VLPs may be produced from influenza virus, however, VLPs may also beproduced from other plasma membrane derived virus including but notlimited to Measles, Ebola, Marburg, and HIV.

The invention includes VLPs of all types of influenza virus which mayinfect humans, including for example, but not limited to the veryprevalent A (H1N1) sub-type (e.g. A/New Caledonia/20/99 (H1N1)), theA/Indonesia/5/05 sub-type (H5N1) (SEQ ID NO: 60) and the less common Btype (for example SEQ ID NO:26, FIG. 10O), and C type (SEQ ID NO:27,FIG. 10P), and to HAs obtained from other influenza subtypes. VLPs ofother influenza subtypes are also included in the present invention, forexample, A/Brisbane/59/2007 (H1N1; SEQ ID NO:48), A/SolomonIslands/3/2006 (H1N1; SEQ ID NO:49), A/Singapore/1/57 (H2N2; SEQ IDNO:54), A/Anhui/1/2005 (H5N1; SEQ ID NO:55), A/Vietnam/1194/2004 (H5N1;SEQ ID NO:56), A/Teal/Hong Kong/W312/97 (H6N1; SEQ ID NO:57), A/HongKong/1073/99 (H9N2; SEQ ID NO:59), A/Brisbane/10/2007 (H3N2; SEQ IDNO:50), A/Wisconsin/67/2005 (H3N2; SEQ ID NO:51), A/Equine/Prague/56(H7N7; SEQ ID NO:58), B/Malaysia/2506/2004 (SEQ ID NO:52), orB/Florida/4/2006 (SEQ ID NO:53).

The present invention also pertains to influenza viruses which infectother mammals or host animals, for example humans, primates, horses,pigs, birds, avian water fowl, migratory birds, quail, duck, geese,poultry, chicken, camel, canine, dogs, feline, cats, tiger, leopard,civet, mink, stone marten, ferrets, house pets, livestock, mice, rats,seal, whale and the like.

Non limiting examples of other antigens that may be expressed in plasmamembrane derived viruses include, the Capsid protein of HIV—p24; gp120,gp41—envelope proteins, the structural proteins VP30 and VP35; Gp/SGP (aglycosylated integral membrane protein) of Filoviruses, for exampleEbola or Marburg, or the H protein, and F protein of Paramyxoviruses,for example, Measles.

The invention also includes, but is not limited to, influenza derivedVLPs that obtain a lipid envelope from the plasma membrane of the cellin which the VLP proteins are expressed. For example, if the VLP isexpressed in a plant-based system, the VLP may obtain a lipid envelopefrom the plasma membrane of the cell.

Generally, the term “lipid” refers to a fat-soluble (lipophilic),naturally-occurring molecules. The term is also used more specificallyto refer to fatty-acids and their derivatives (including tri-, di-, andmonoglycerides and phospholipids), as well as other fat-solublesterol-containing metabolites or sterols. Phospholipids are a majorcomponent of all biological membranes, along with glycolipids, sterolsand proteins. Examples of phospholipids includephosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol,phosphatidylserine, phosphatidylglycerol and the like. Examples ofsterols include zoosterols (for example, cholesterol) and phytosterols(for example, sitosterol) and steryl-glucoside. Over 200 phytosterolshave been identified in various plant species, the most common beingcampesterol, stigmasterol, ergosterol, brassicasterol,delta-7-stigmasterol, delta-7-avenasterol, daunosterol, sitosterol,24-methylcholesterol, cholesterol or beta-sitosterol. As one of skill inthe art would understand, the lipid composition of the plasma membraneof a cell may vary with the culture or growth conditions of the cell ororganism from which the cell is obtained.

Cell membranes generally comprise lipid bilayers, as well as proteinsfor various functions. Localized concentrations of particular lipids maybe found in the lipid bilayer, referred to as ‘lipid rafts’. Withoutwishing to be bound by theory, lipid rafts may have significant roles inendo and exocytosis, entry or egress of viruses or other infectiousagents, inter-cell signal transduction, interaction with otherstructural components of the cell or organism, such as intracellular andextracellular matrices.

With reference to influenza virus, the term “hemagglutinin” or “HA” asused herein refers to a glycoprotein found on the outside of influenzaviral particles. HA is a homotrimeric membrane type I glycoprotein,generally comprising a signal peptide, an HA1 domain, and an HA2 domaincomprising a membrane-spanning anchor site at the C-terminus and a smallcytoplasmic tail (FIG. 1B). Nucleotide sequences encoding HA are wellknown and are available—see, for example, the BioDefence Public Healthbase (Influenza Virus; see URL: biohealthbase.org) or National Centerfor Biotechnology Information (see URL: ncbi.nlm.nih.gov), both of whichare incorporated herein by reference.

The term “homotrimer” or “homotrimeric” indicates that an oligomer isformed by three HA protein molecules. Without wishing to be bound bytheory, HA protein is synthesized as monomeric precursor protein (HA0)of about 75 kDa, which assembles at the surface into an elongatedtrimeric protein. Before trimerization occurs, the precursor protein iscleaved at a conserved activation cleavage site (also referred to asfusion peptide) into 2 polypeptide chains, HA1 and HA2 (comprising thetransmembrane region), linked by a disulfide bond. The HA1 segment maybe 328 amino acids in length, and the HA2 segment may be 221 amino acidsin length. Although this cleavage may be important for virusinfectivity, it may not be essential for the trimerization of theprotein. Insertion of HA within the endoplasmic reticulum (ER) membraneof the host cell, signal peptide cleavage and protein glycosylation areco-translational events. Correct refolding of HA requires glycosylationof the protein and formation of 6 intra-chain disulfide bonds. The HAtrimer assembles within the cis- and trans-Golgi complex, thetransmembrane domain playing a role in the trimerization process. Thecrystal structures of bromelain-treated HA proteins, which lack thetransmembrane domain, have shown a highly conserved structure amongstinfluenza strains. It has also been established that HA undergoes majorconformational changes during the infection process, which requires theprecursor HA0 to be cleaved into the 2 polypeptide chains HA1 and HA2.The HA protein may be processed (i.e., comprise HA1 and HA2 domains), ormay be unprocessed (i.e. comprise the HA0 domain).

The present invention pertains to the use of an HA protein comprisingthe transmembrane domain and includes HA1 and HA2 domains, for examplethe HA protein may be HA0, or processed HA comprising HA1 and HA2. TheHA protein may be used in the production or formation of VLPs using aplant, or plant cell, expression system.

The HA of the present invention may be obtained from any subtype. Forexample, the HA may be of subtype H1, H2, H3, H4, H5, H6, H7, H8, H9,H10, H11, H12, H13, H14, H15, H16 or of influenza type B. Therecombinant HA of the present invention may also comprise an amino acidsequence based on the sequence any hemagglutinin known in the art-see,for example, the BioDefence Public Health base (Influenza Virus; seeURL: biohealthbase.org) or National Center for Biotechnology Information(see URL: ncbi.nlm.nih.gov). Furthermore, the HA may be based on thesequence of a hemagglutinin that is isolated from one or more emergingor newly-identified influenza viruses.

The present invention also includes VLPs that comprise HAs obtained fromone or more than one influenza subtype. For example, VLPs may compriseone or more than one HA from the subtype H1 (encoded by SEQ ID NO:28),H2 (encoded by SEQ ID NO:12), H3 (encoded by SEQ ID NO:13), H4 (encodedby SEQ ID NO:14), H5 (encoded by SEQ ID NO:15), H6 (encoded by SEQ IDNO:16), H7 (encoded by SEQ ID NO:11), H8 (encoded by SEQ ID NO:17), H9(encoded by SEQ ID NO:18), H10 (encoded by SEQ ID NO:19), H11 (encodedby SEQ ID NO:20), H12 (encoded by SEQ ID NO:21), H13 (encoded by SEQ IDNO:27), H14 (encoded by SEQ ID NO:23), H15 (encoded by SEQ ID NO:24),H16 (encoded by SEQ ID NO:25), or influenza type B (encoded by SEQ IDNO: 26), or a combination thereof. One or more that one HA from the oneor more than one influenza subtypes may be co-expressed within a plantor insect cell to ensure that the synthesis of the one or more than oneHA results in the formation of VLPs comprising a combination of HAsobtained from one or more than one influenza subtype. Selection of thecombination of HAs may be determined by the intended use of the vaccineprepared from the VLP. For example a vaccine for use in inoculatingbirds may comprise any combination of HA subtypes, while VLPs useful forinoculating humans may comprise subtypes one or more than one ofsubtypes H1, H2, H3, H5, H7, H9, H10, N1, N2, N3 and N7. However, otherHA subtype combinations may be prepared depending upon the use of theinoculum.

Therefore, the present invention is directed to a VLP comprising one ormore than one HA subtype, for example two, three, four, five, six, ormore HA subtypes.

The present invention also provides for nucleic acids encodinghemagglutinins that form VLPs when expressed in plants.

Exemplary nucleic acids may comprise nucleotide sequences ofhemagglutinin from selected strains of influenza subtypes. For example,an A (H1N1) sub-type such as A/New Caledonia/20/99 (H1N1) (SEQ ID NO:33), the A/Indonesia/5/05 sub-type (H5N1) (comprising construct #660;SEQ ID NO: 60) and the less common B type (for example SEQ ID NO:26,FIG. 10O), and C type (SEQ ID NO:27, FIG. 10P), and to HAs obtained fromother influenza subtypes. VLPs of other influenza subtypes are alsoincluded in the present invention, for example, A/Brisbane/59/2007(H1N1; SEQ ID NO:36), A/Solomon Islands/3/2006 (H1N1; SEQ ID NO:37),A/Singapore/1/57 (H2N2; SEQ ID NO:42), A/Anhui/1/2005 (H5N1; SEQ IDNO:43), A/Vietnam/1194/2004 (H5N1; SEQ ID NO:44), A/Teal/HongKong/W312/97 (H6N1; SEQ ID NO:45), A/Hong Kong/1073/99 (H9N2; SEQ IDNO:47), A/Brisbane/10/2007 (H3N2; SEQ ID NO:38), A/Wisconsin/67/2005(H3N2; SEQ ID NO:39), A/Equine/Prague/56 (H7N7; SEQ ID NO:46),B/Malaysia/2506/2004 (SEQ ID NO:40), or B/Florida/4/2006 (SEQ ID NO:41).

Correct folding of the hemagglutinins may be important for stability ofthe protein, formation of multimers, formation of VLPs and function ofthe HA (ability to hemagglutinate), among other characteristics ofinfluenza hemagglutinins. Folding of a protein may be influenced by oneor more factors, including, but not limited to, the sequence of theprotein, the relative abundance of the protein, the degree ofintracellular crowding, the availability of cofactors that may bind orbe transiently associated with the folded, partially folded or unfoldedprotein, the presence of one or more chaperone proteins, or the like.

Heat shock proteins (Hsp) or stress proteins are examples of chaperoneproteins, which may participate in various cellular processes includingprotein synthesis, intracellular trafficking, prevention of misfolding,prevention of protein aggregation, assembly and disassembly of proteincomplexes, protein folding, and protein disaggregation. Examples of suchchaperone proteins include, but are not limited to, Hsp60, Hsp65, Hsp70, Hsp90, Hsp100, Hsp20-30, Hsp10, Hsp100-200, Hsp100, Hsp90, Lon,TF55, FKBPs, cyclophilins, ClpP, GrpE, ubiquitin, calnexin, and proteindisulfide isomerases. See, for example, Macario, A. J. L., Cold SpringHarbor Laboratory Res. 25:59-70. 1995; Parsell, D. A. & Lindquist, S.Ann. Rev. Genet. 27:437-496 (1993); U.S. Pat. No. 5,232,833. In someexamples, a particular group of chaperone proteins includes Hsp40 andHsp70.

Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaKfrom bacteria, particularly mycobacteria such as Mycobacterium leprae,Mycobacterium tuberculosis, and Mycobacterium bovis (such asBacille-Calmette Guerin: referred to herein as Hsp71). DnaK fromEscherichia coli, yeast. and other prokaryotes, and BiP and Grp78 fromeukaryotes, such as A. thaliana (Lin et al. 2001 (Cell Stress andChaperones 6:201-208). A particular example of an Hsp70 is A. thalianaHsp70 (encoded by SEQ ID NO: 122, or SEQ ID NO: 123). Hsp70 is capableof specifically binding ATP as well as unfolded polypeptides andpeptides, thereby participating in protein folding and unfolding as wellas in the assembly and disassembly of protein complexes.

Examples of Hsp40 include DnaJ from prokaryotes such as E. coli andmycobacteria and HSJ1, HDJ1 and Hsp40 from eukaryotes, such as alfalfa(Frugis et al., 1999. Plant Molecular Biology 40:397-408). A particularexample of an Hsp40 is M. sativa MsJ1 (encoded by SEQ ID NO: 121, 123 or114). Hsp40 plays a role as a molecular chaperone in protein folding,thermotolerance and DNA replication, among other cellular activities.

Among Hsps, Hsp70 and its co-chaperone, Hsp40, are involved in thestabilization of translating and newly synthesized polypeptides beforethe synthesis is complete. Without wishing to be bound by theory, Hsp40binds to the hydrophobic patches of unfolded (nascent or newlytransferred) polypeptides, thus facilitating the interaction ofHsp70-ATP complex with the polypeptide. ATP hydrolysis leads to theformation of a stable complex between the polypeptide, Hsp70 and ADP,and release of Hsp40. The association of Hsp70-ADP complex with thehydrophobic patches of the polypeptide prevents their interaction withother hydrophobic patches, preventing the incorrect folding and theformation of aggregates with other proteins (reviewed in Hartl, F U.1996. Nature 381:571-579).

Again, without wishing to be bound by theory, as protein productionincreases in a recombinant protein expression system, the effects ofcrowding on recombinant protein expression may result in aggregationand/or reduced accumulation of the recombinant protein resulting fromdegradation of misfolded polypeptide. Native chaperone proteins may beable to facilitate correct folding of low levels of recombinant protein,but as the expression levels increase, native chaperones may become alimiting factor. High levels of expression of hemagglutinin in theagroinfiltrated leaves may lead to the accumulation of hemagglutininpolypeptides in the cytosol, and co-expression of one or more than onechaperone proteins such as Hsp70, Hsp40 or both Hsp70 and Hsp40 mayincrease stability in the cytosol of the cells expressing thepolypeptides cells, thus reducing the level of misfolded or aggregatedhemagglutinin polypeptides, and increasing the number of polypeptidesaccumulate as stable hemagglutinin, exhibiting tertiary and quaternarystructural characteristics that allow for hemagglutination and/orformation of virus-like particles.

Therefore, the present invention also provides for a method of producinginfluenza VLPs in a plant, wherein a first nucleic acid encoding aninfluenza HA is co-expressed with a second nucleic acid encoding achaperone. The first and second nucleic acids may be introduced to theplant in the same step, or may be introduced to the plant sequentially.The present invention also provides for a method of producing influenzaVLPs in a plant, where the plant comprises the first nucleic acid, andthe second nucleic acid is subsequently introduced.

The present invention also provides for a plant comprising a nucleicacid encoding one, or more than one influenza hemagglutinin and anucleic acid encoding one or more than one chaperones.

Processing of an N-terminal signal peptide (SP) sequence duringexpression and/or secretion of influenza hemagglutinins has beenproposed to have a role in the folding process. The term “signalpeptide” refers generally to a short (about 5-30 amino acids) sequenceof amino acids, found generally at the N-terminus of a hemagglutininpolypeptide that may direct translocation of the newly-translatedpolypeptide to a particular organelle, or aid in positioning of specificdomains of the polypeptide. The signal peptide of hemagglutinins targetthe translocation of the protein into the endoplasmic reticulum and havebeen proposed to aid in positioning of the N-terminus proximal domainrelative to a membrane-anchor domain of the nascent hemagglutininpolypeptide to aid in cleavage and folding of the mature hemagglutinin.Removal of a signal peptide (for example, by a signal peptidase), mayrequire precise cleavage and removal of the signal peptide to providethe mature hemagglutinin—this precise cleavage may be dependent on anyof several factors, including a portion or all of the signal peptide,amino acid sequence flanking the cleavage site, the length of the signalpeptide, or a combination of these, and not all factors may apply to anygiven sequence.

A signal peptide may be native to the hemagglutinin being expressed, ora recombinant hemagglutinin comprising a signal peptide from a firstinfluenza type, subtype or strain with the balance of the hemagglutininfrom a second influenza type, subtype or strain. For example the nativeSP of HA subtypes H1, H2, H3, H5, H6, H7, H9 or influenza type B may beused to express the HA in a plant system.

A signal peptide may also be non-native, for example, from a structuralprotein or hemagglutinin of a virus other than influenza, or from aplant, animal or bacterial polypeptide. An exemplary signal peptide isthat of alfalfa protein disulfide isomerase (PDISP) (nucleotides 32-103of Accession No. Z11499; SEQ ID NO: 34; FIG. 17; amino acid sequenceMAKNVAIFGLLFSLLLLVPSQIFAEE).

The present invention also provides for an influenza hemagglutinincomprising a native, or a non-native signal peptide, and nucleic acidsencoding such hemagglutinins.

Influenza HA proteins exhibit a range of similarities and differenceswith respect to molecular weight, isoelectric point, size, glycancomplement and the like. The physico-chemical properties of the varioushemagglutinins may be useful to allow for differentiation between theHAs expressed in a plant, insect cell or yeast system, and may be ofparticular use when more than one HA is co-expressed in a single system.Examples of such physico-chemical properties are provided in Table 1.

TABLE 1 Physico-chemical properties of influenza hemagglutinins Clone AAGlycans Molecular Weight (kDA) Isoelectric point No Type Influenzastrains HA0 HA1 HA2 HA0 HA1 HA2 HA0 HA0¹ HA1 HA1¹ HA2 HA2¹ HA0 HA1 HA2774 H1 A/Brisbane/59/2007 548 326 222 9 7 2 61 75 36 47 25 28 6.4 7.55.3 775 H1 A/Solomon Islands/ 548 326 222 9 7 2 61 75 36 47 25 28 6.16.7 5.3 3/2006 776 H3 A/Brisbane/10/2007 550 329 221 12 11 1 62 80 37 5425 27 8.5 9.6 5.2 777 H3 A/Wisconsin/67/2005 550 329 221 11 10 1 62 7937 52 25 27 8.8 9.6 5.3 778 B B/Malaysia/2506/2004 570 347 223 12 8 4 6280 38 50 24 30 8.0 9.7 4.5 779 B B/Florida/4/2006 569 346 223 10 7 3 6277 38 48 24 29 8.0 9.7 4.5 780 H2 A/Singapore/1/57 547 325 222 6 4 2 6271 36 42 25 28 6.0 7.5 4.9 781 H5 A/Anhui/1/2005 551 329 222 7 5 2 62 7337 45 25 28 6.2 8.9 4.7 782 H5 A/Vietnam/1194/2004 552 330 222 7 5 2 6374 38 45 25 28 6.4 9.1 4.8 783 H6 A/Teal/Hong Kong/ 550 328 222 8 5 3 6275 37 45 25 30 5.7 5.9 5.6 W312/97 784 H7 A/Equine/Prague/56 552 331 2216 4 2 62 71 37 43 25 28 8.9 9.7 4.9 785 H9 A/Hong Kong/ 542 320 199 9 72 61 75 36 46 23 26 8.4 9.5 5.3 1073/99

The present invention also includes nucleotide sequences SEQ ID NO:28;SEQ ID NO:3; SEQ ID NO:11, encoding HA from H1, H5 or H7, respectively.The present invention also includes a nucleotide sequence thathybridizes under stringent hybridisation conditions to SEQ ID NO:28; SEQID NO:3; SEQ ID NO:11. The present invention also includes a nucleotidesequence that hybridizes under stringent hybridisation conditions to acompliment of SEQ ID NO:28; SEQ ID NO:3; SEQ ID NO:1. These nucleotidesequences that hybridize to SEQ ID or a complement of SEQ ID encode ahemagglutinin protein that, when expressed forms a VLP, and the VLPinduces production of an antibody when administered to a subject. Forexample, expression of the nucleotide sequence within a plant cell formsa VLP, and the VLP may be used to produce an antibody that is capable ofbinding HA, including mature HA, HA0, HA1, or HA2 of one or moreinfluenza types or subtypes. The VLP, when administered to a subject,induces an immune response.

The present invention also includes nucleotide sequences SEQ ID NO:12SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO:27, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47. Thepresent invention also includes a nucleotide sequence that hybridizesunder stringent hybridisation conditions to SEQ ID NO:12 SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47. The present inventionalso includes a nucleotide sequence that hybridizes under stringenthybridisation conditions to a compliment of SEQ ID NO:12 SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47. These nucleotidesequences that hybridize to SEQ ID NO:12 SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46 or SEQ ID NO:47 or a complement of SEQ ID NO:12 SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO:27, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47 encode ahemagglutinin protein that, when expressed forms a VLP, and the VLPinduces production of an antibody when administered to a subject. Forexample, expression of the nucleotide sequence within a plant cell formsa VLP, and the VLP may be used to produce an antibody that is capable ofbinding HA, including mature HA, HA0, HAL or HA2 of one or moreinfluenza types or subtypes. The VLP, when administered to a subject,induces an immune response.

In some embodiments, the present invention also includes nucleotidesequences SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47,encoding HA from H1, H2, H3, H5, H7 or H9 subtypes of influenza A, or HAfrom type B influenza. The present invention also includes a nucleotidesequence that hybridizes under stringent hybridisation conditions to SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47. The present inventionalso includes a nucleotide sequence that hybridizes under stringenthybridisation conditions to a compliment of SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46 or SEQ ID NO:47. These nucleotide sequences thathybridize to SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47or a complement of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ IDNO:47 encode a hemagglutinin protein that, when expressed forms a VLP,and the VLP induces production of an antibody when administered to asubject. For example, expression of the nucleotide sequence within aplant cell forms a VLP, and the VLP may be used to produce an antibodythat is capable of binding HA, including mature HA, HA0, HA1, or HA2 ofone or more influenza types or subtypes. The VLP, when administered to asubject, induces an immune response.

Hybridization under stringent hybridization conditions is known in theart (see for example Current Protocols in Molecular Biology, Ausubel etal., eds. 1995 and supplements; Maniatis et al., in Molecular Cloning (ALaboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook andRussell, in Molecular Cloning: A Laboratory Manual, 3^(rd) edition 2001;each of which is incorporated herein by reference). An example of onesuch stringent hybridization conditions may be about 16-20 hourshybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65°C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30minutes. Alternatively, an exemplary stringent hybridization conditioncould be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C.,followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours),or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO₄buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in2×SSC, 0.1% SDS for 20 or 30 minutes each.

Additionally, the present invention includes nucleotide sequences thatare characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequenceidentity, or sequence similarity, with the nucleotide sequence encodingHA from H1 (SEQ ID NO:28), H5 (SEQ ID NO:3) or H7 (SEQ ID NO:11),wherein the nucleotide sequence encodes a hemagglutinin protein thatwhen expressed forms a VLP, and that the VLP induces the production ofan antibody. For example, expression of the nucleotide sequence within aplant cell forms a VLP, and the VLP may be used to produce an antibodythat is capable of binding HA, including mature HA, HA0, HA1, or HA2.The VLP, when administered to a subject, induces an immune response.

Additionally, the present invention includes nucleotide sequences thatare characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequenceidentity, or sequence similarity, with the nucleotide sequence of SEQ IDNO:12 SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO:27, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47,wherein the nucleotide sequence encodes a hemagglutinin protein thatwhen expressed forms a VLP, and that the VLP induces the production ofan antibody. For example, expression of the nucleotide sequence within aplant cell forms a VLP, and the VLP may be used to produce an antibodythat is capable of binding HA, including mature HA, HA0, HA1, or HA2.The VLP, when administered to a subject, induces an immune response.

Additionally, the present invention includes nucleotide sequences thatare characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequenceidentity, or sequence similarity, with the nucleotide sequence of SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, wherein thenucleotide sequence encodes a hemagglutinin protein that when expressedforms a VLP, and that the VLP induces the production of an antibody. Forexample, expression of the nucleotide sequence within a plant cell formsa VLP, and the VLP may be used to produce an antibody that is capable ofbinding HA, including mature HA, HA0, HA1, or HA2. The VLP, whenadministered to a subject, induces an immune response.

Similarly, the present invention includes HAs associated with thefollowing subtypes H1 (encoded by SEQ ID NO:28), H2 (encoded by SEQ IDNO:12), H3 (encoded by SEQ ID NO:13), H4 (encoded by SEQ ID NO:14), H5(encoded by SEQ ID NO:15), 1-16 (encoded by SEQ ID NO:16), H7 (encodedby SEQ ID NO:11), H8 (encoded by SEQ ID NO:17), H9 (encoded by SEQ IDNO:18), H10 (encoded by SEQ ID NO:19), H11 (encoded by SEQ ID NO:20),H12 (encoded by SEQ ID NO:21), H13 (encoded by SEQ ID NO:27), H14(encoded by SEQ ID NO:23), H15 (encoded by SEQ ID NO:24), H16 (encodedby SEQ ID NO:25), or influenza type B (encoded by SEQ ID NO: 26); seeFIGS. 10A to 10O), and nucleotide sequences that are characterized ashaving from about 70 to 100% or any amount therebetween, 80 to 100% orany amount there between, 90-100% or any amount therebetween, or 95-100%or any amount therebetween, sequence identity with H1 (SEQ ID NO:28), H2(SEQ ID NO:12), H3 (SEQ ID NO:13), H4 (SEQ ID NO:14), H5 (SEQ ID NO:15),H6 (SEQ ID NO:16), H7 (SEQ ID NO:11), H8 (SEQ ID NO:17), H9 (SEQ IDNO:18), H10 (SEQ ID NO:19), H11 (SEQ ID NO:20), H12 (SEQ ID NO:21), H13(SEQ ID NO:27), H14 (SEQ ID NO:23), H15 (SEQ ID NO:24), H16 (SEQ IDNO:25), wherein the nucleotide sequence encodes a hemagglutinin proteinthat when expressed forms a VLP, and that the VLP induces the productionof an antibody. For example, expression of the nucleotide sequencewithin a plant cell forms a VLP, and the VLP may be used to produce anantibody that is capable of binding HA, including mature HA, HA0, HA1,or HA2. The VLP, when administered to a subject, induces an immuneresponse.

An “immune response” generally refers to a response of the adaptiveimmune system. The adaptive immune system generally comprises a humoralresponse, and a cell-mediated response. The humoral response is theaspect of immunity that is mediated by secreted antibodies, produced inthe cells of the B lymphocyte lineage (B cell). Secreted antibodies bindto antigens on the surfaces of invading microbes (such as viruses orbacteria), which flags them for destruction. Humoral immunity is usedgenerally to refer to antibody production and the processes thataccompany it, as well as the effector functions of antibodies, includingTh2 cell activation and cytokine production, memory cell generation,opsonin promotion of phagocytosis, pathogen elimination and the like.The terms “modulate” or “modulation” or the like refer to an increase ordecrease in a particular response or parameter, as determined by any ofseveral assays generally known or used, some of which are exemplifiedherein.

A cell-mediated response is an immune response that does not involveantibodies but rather involves the activation of macrophages, naturalkiller cells (NK), antigen-specific cytotoxic T-lymphocytes, and therelease of various cytokines in response to an antigen. Cell-mediatedimmunity is used generally to refer to some Th cell activation, Tc cellactivation and T-cell mediated responses. Cell mediated immunity is ofparticular importance in responding to viral infections.

For example, the induction of antigen specific CD8 positive Tlymphocytes may be measured using an ELISPOT assay; stimulation of CD4positive T-lymphocytes may be measured using a proliferation assay.Anti-influenza antibody titres may be quantified using an ELISA assay;isotypes of antigen-specific or cross reactive antibodies may also bemeasured using anti-isotype antibodies (e.g. anti-IgG, IgA, IgE or IgM).Methods and techniques for performing such assays are well-known in theart.

A hemagglutination inhibition (HI, or HAI) assay may also be used todemonstrate the efficacy of antibodies induced by a vaccine, or vaccinecomposition can inhibit the agglutination of red blood cells (RBC) byrecombinant HA. Hemagglutination inhibitory antibody titers of serumsamples may be evaluated by microtiter HAI (Aymard et al 1973).Erythrocytes from any of several species may be used—e.g. horse, turkey,chicken or the like. This assay gives indirect information on assemblyof the HA trimer on the surface of VLP, confirming the properpresentation of antigenic sites on HAs.

Cross-reactivity HAI titres may also be used to demonstrate the efficacyof an immune response to other strains of virus related to the vaccinesubtype. For example, serum from a subject immunized with a vaccinecomposition of a first strain (e.g. VLPs of A/Indonesia 5/05) may beused in an HAI assay with a second strain of whole virus or virusparticles (e.g. A/Vietnam/1194/2004), and the HAI titer determined.

Cytokine presence or levels may also be quantified. For example aT-helper cell response (Th1/Th2) will be characterized by themeasurement of IFN-γ and IL-4 secreting cells using by ELISA (e.g. BDBiosciences OptEIA kits). Peripheral blood mononuclear cells (PBMC) orsplenocytes obtained from a subject may be cultured, and the supernatantanalyzed. T lymphocytes may also be quantified by fluorescence-activatedcell sorting (FACS), using marker specific fluorescent labels andmethods as are known in the art.

A microneutralization assay may also be conducted to characterize animmune response in a subject, see for example the methods of Rowe etal., 1973. Virus neutralization titers may be obtained several ways,including: 1) enumeration of lysis plaques (plaque assay) followingcrystal violet fixation/coloration of cells; 2) microscopic observationof cell lysis in culture; 3) ELISA and spectrophotometric detection ofNP virus protein (correlate with virus infection of host cells).

Sequence identity or sequence similarity may be determined using anucleotide sequence comparison program, such as that provided withinDNASIS (for example, using, but not limited to, the followingparameters: GAP penalty 5, # of top diagonals 5, fixed GAP penalty 10,k-tuple 2, floating gap 10, and window size 5). However, other methodsof alignment of sequences for comparison are well-known in the art forexample the algorithms of Smith & Waterman (1981, Adv. Appl. Math.2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson &Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerizedimplementations of these algorithms (e.g. GAP, BESTFIT, FASTA, andBLAST), or by manual alignment and visual inspection.

The term “hemagglutinin domain” refers to a peptide comprising eitherthe HA0 domain, or the HA1 and HA2 domains (alternately referred to asHA1 and HA2 fragments). HA0 is a precursor of the HA1 and HA2 fragments.The HA monomer may be generally subdivided in 2 functional domains—thestem domain and the globular head, or head domain. The stem domain isinvolved in infectivity and pathogenicity of the virus via theconformational change it may undergo when exposed to acidic pH. The stemdomain may be further subdivided into 4 subdomains or fragments—thefusion sub-domain or peptide (a hydrophobic stretch of amino acidsinvolved in fusion with the host membrane in the acidic pHconformational state); the stem sub-domain (may accommodate the two ormore conformations), the transmembrane domain or sub-domain (TmD)(involved in the affinity of the HA for lipid rafts), and thecytoplasmic tail (cytoplasmic tail sub-domain) (Ctail) (involved insecretion of HA). The globular head is divided in 2 subdomains, the RBsubdomain and the vestigial esterase domain (E). The E subdomain may bepartially or fully buried and not exposed at the surface of the globularhead, thus some antibodies raised against HA bind to the RB subdomain.

The term “virus like particle” (VLP), or “virus-like particles” or“VLPs” refers to structures that self-assemble and comprise structuralproteins such as influenza HA protein. VLPs are generallymorphologically and antigenically similar to virions produced in aninfection, but lack genetic information sufficient to replicate and thusare non-infectious. In some examples, VLPs may comprise a single proteinspecies, or more than one protein species. For VLPs comprising more thanone protein species, the protein species may be from the same species ofvirus, or may comprise a protein from a different species, genus,subfamily or family of virus (as designated by the ICTV nomenclature).In other examples, one or more of the protein species comprising a VLPmay be modified from the naturally occurring sequence. VLPs may beproduced in suitable host cells including plant and insect host cells.Following extraction from the host cell and upon isolation and furtherpurification under suitable conditions, VLPs may be purified as intactstructures.

The VLPs produced from influenza derived proteins, in accordance withthe present invention do not comprise M1 protein. The M1 protein isknown to bind RNA (Wakefield and Brownlee, 1989) which is a contaminantof the VLP preparation. The presence of RNA is undesired when obtainingregulatory approval for the VLP product, therefore a VLP preparationlacking RNA may be advantageous.

The VLPs of the present invention may be produced in a host cell that ischaracterized by lacking the ability to sialylate proteins, for examplea plant cell, an insect cell, fungi, and other organisms includingsponge, coelenterara, annelida, arthoropoda, mollusca, nemathelminthea,trochelmintes, plathelminthes, chaetognatha, tentaculate, chlamydia,spirochetes, gram-positive bacteria, cyanobacteria, archaebacteria, orthe like. See, for example Glycoforum (URL:glycoforum.gr.jp/science/word/evolution/ES-A03E.html) or Gupta et al.,1999. Nucleic Acids Research 27:370-372; or Toukach et al., 2007.Nucleic Acids Research 35:D280-D286; or URL: glycostructures.jp(Nakahara et al., 2008. Nucleic Acids Research 36:D368-D371; publishedonline Oct. 11, 2007 doi:10.1093/NAR/gkm=833). The VLPs produced asdescribed herein do not typically comprise neuramindase (NA). However,NA may be co-expressed with HA should VLPs comprising HA and NA bedesired.

A VLP produced in a plant according to some aspects of the invention maybe complexed with plant-derived lipids. The VLP may comprise an HA0, HA1or HA2 peptide. The plant-derived lipids may be in the form of a lipidbilayer, and may further comprise an envelope surrounding the VLP. Theplant derived lipids may comprise lipid components of the plasmamembrane of the plant where the VLP is produced, including, but notlimited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE),glycosphingolipids, phytosterols or a combination thereof. Aplant-derived lipid may alternately be referred to as a ‘plant lipid’.Examples of phytosterols are known in the art, and include, for example,stigmasterol, sitosterol, 24-methylcholesterol and cholesterol—see, forexample, Mongrand et al., 2004.

VLPs may be assessed for structure and size by, for example,hemagglutination assay, electron microscopy, or by size exclusionchromatography.

For size exclusion chromatography, total soluble proteins may beextracted from plant tissue by homogenizing (Polytron) sample offrozen-crushed plant material in extraction buffer, and insolublematerial removed by centrifugation. Precipitation with PEG may also beof benefit. The soluble protein is quantified, and the extract passedthrough a Sephacryl™ column. Blue Dextran 2000 may be used as acalibration standard. Following chromatography, fractions may be furtheranalyzed by immunoblot to determine the protein complement of thefraction.

Without wishing to be bound by theory, the capacity of HA to bind to RBCfrom different animals is driven by the affinity of HA for sialic acidsα2,3 or α2,3 and the presence of these sialic acids on the surface ofRBC. Equine and avian HA from influenza viruses agglutinate erythrocytesfrom all several species, including turkeys, chickens, ducks, guineapigs, humans, sheep, horses and cows; whereas human HAs will bind toerythrocytes of turkey, chickens, ducks, guina pigs, humans and sheep(see also Ito T. et al, 1997, Virology, vol 227, p493-499; and MedeirosR et al, 2001, Virology, vol 289 p. 74-85). Examples of the speciesreactivity of HAs of different influenza strains is shown in Tables 2Aand 2B.

TABLE 2A Species of RBC bound by HAs of selected seasonal influenzastrains. Sea- sonal Strain No Origin Horse Turkey H1 A/Brisbane/59/2007(H1N1) 774 Human + ++ A/Solomon Islands/3/2006 775 Human + ++ (H1N1) H3A/Brisbane/10/2007 (H3N2) 776 Human + ++ A/Wisconsin/67/2005 (H3N2) 777Human + ++ B B/Malaysia/2506/2004 778 Human + ++ B/Florida/4/2006 779Human + ++

TABLE 2B Species of RBC bound by HAs of selected pandemic influenzastrains Pan- Tur- demic Strain No Origin Horse key H2 A/Singapore/1/57(H2N2) 780 Human + ++ H5 A/Anhui/1/2005 (H5N1) 781 Hu-Av ++ +A/Vietnam/1194/2004 (H5N1) 782 Hu-Av ++ + H6 A/Teal/Hong Kong/W312/97783 Avian ++ + (H6N1) H7 A/Equine/Prague/56 (H7N7) 784 Equine ++ ++ H9A/Hong Kong/1073/99 (H9N2) 785 Human ++ +

A fragment or portion of a protein, fusion protein or polypeptideincludes a peptide or polypeptide comprising a subset of the amino acidcomplement of a particular protein or polypeptide, provided that thefragment can form a VLP when expressed. The fragment may, for example,comprise an antigenic region, a stress-response-inducing region, or aregion comprising a functional domain of the protein or polypeptide. Thefragment may also comprise a region or domain common to proteins of thesame general family, or the fragment may include sufficient amino acidsequence to specifically identify the full-length protein from which itis derived.

For example, a fragment or portion may comprise from about 60% to about100%, of the length of the full length of the protein, or any amounttherebetween, provided that the fragment can form a VLP when expressed.For example, from about 60% to about 100%, from about 70% to about 100%,from about 80% to about 100%, from about 90% to about 100%, from about95% to about 100%, of the length of the full length of the protein, orany amount therebetween. Alternately, a fragment or portion may be fromabout 150 to about 500 amino acids, or any amount therebetween,depending upon the HA, and provided that the fragment can form a VLPwhen expressed. For example, a fragment may be from 150 to about 500amino acids, or any amount therebetween, from about 200 to about 500amino acids, or any amount therebetween, from about 250 to about 500amino acids, or any amount therebetween, from about 300 to about 500 orany amount therebetween, from about 350 to about 500 amino acids, or anyamount therebetween, from about 400 to about 500 or any amounttherebetween, from about 450 to about 500 or any amount therebetween,depending upon the HA, and provided that the fragment can form a VLPwhen expressed. For example, about 5, 10, 20, 30, 40 or 50 amino acids,or any amount therebetween may be removed from the C terminus, the Nterminus or both the N and C terminus of an HA protein, provided thatthe fragment can form a VLP when expressed.

Numbering of amino acids in any given sequence are relative to theparticular sequence, however one of skill can readily determine the‘equivalency’ of a particular amino acid in a sequence based onstructure and/or sequence. For example, if 6 N terminal amino acids wereremoved when constructing a clone for crystallography, this would changethe specific numerical identity of the amino acid (e.g. relative to thefull length of the protein), but would not alter the relative positionof the amino acid in the structure.

Comparisons of a sequence or sequences may be done using a BLASTalgorithm (Altschul et al., 1990. J. Mol. Biol 215:403-410). A BLASTsearch allows for comparison of a query sequence with a specificsequence or group of sequences, or with a larger library or database(e.g. GenBank or GenPept) of sequences, and identify not only sequencesthat exhibit 100% identity, but also those with lesser degrees ofidentity. Nucleic acid or amino acid sequences may be compared using aBLAST algorithm. Furthermore the identity between two or more sequencesmay be determined by aligning the sequences together and determining the% identity between the sequences. Alignment may be carried out using theBLAST Algorithm (for example as available through GenBank; URL:ncbi.nlm.nih.gov/cgi-bin/BLAST/ using default parameters: Program:blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise;Query genetic Codes: Standard(1)), or BLAST2 through EMBL URL:embl-heidelberg.de/Services/index.html using default parameters: MatrixBLOSUM62; Filter: default, echofilter: on, Expect: 10, cutoff: default;Strand: both; Descriptions: 50, Alignments: 50; or FASTA, using defaultparameters), or by manually comparing the sequences and calculating the% identity.

The present invention describes, but is not limited to, the cloning of anucleic acid encoding HA into a plant expression vector, and theproduction of influenza VLPs from the plant, suitable for vaccineproduction. Examples of such nucleic acids include, for example, but arenot limited to, an influenza A/New Caledonia/20/99 (H1N1) virus HA (e.g.SEQ ID NO: 61), an HA from A/Indonesia/5/05 sub-type (H5N1) (e.g. SEQ IDNO: 60), A/Brisbane/59/2007 (H1N1) (e.g. SEQ ID NO: 36, 48, 62),A/Solomon Islands/3/2006 (H1N1) (e.g. SEQ ID NO: 37, 49, 63),A/Singapore/1/57 (H2N2) (e.g. SEQ ID NO: 42, 54, 64), A/Anhui/1/2005(H5N1) (e.g. SEQ ID NO: 43, 55, 65), A/Vietnam/1194/2004 (H5N1) (e.g.SEQ ID NO: 44, 56, 66), A/Teal/Hong Kong/W312/97 (H6N1) (e.g. SEQ ID NO:45, 57, 67), A/Hong Kong/1073/99 (H9N2) (e.g. SEQ ID NO: 47, 59, 68),A/Brisbane/10/2007 (H3N2) (e.g. SEQ ID NO: 38, 50, 69),A/Wisconsin/67/2005 (H3N2) (e.g. SEQ ID NO: 39, 51, 70),A/Equine/Prague/56 (H7N7) (e.g. SEQ ID NO: 46, 58, 71),B/Malaysia/2506/2004 (e.g. SEQ ID NO: 40, 52, 72), B/Florida/4/2006(e.g. SEQ ID NO: 41, 53, 73). The corresponding clone or constructnumbers for these strains is provided in Table 1. Nucleic acid sequencescorresponding to SEQ ID NOs: 36-47 comprise a plastocyanin upstream andoperatively linked to the coding sequence of the HA for each of thetypes or subtypes, as illustrated in FIGS. 28-39. Nucleic acid sequencescorresponding to SEQ ID NO: 60-73 comprise an HA expression cassettecomprising alfalfa plastocyanin promoter and 5′ UTR, hemagglutinincoding sequence of an HA, alfalfa plastocyanin 3′ UTR and terminatorsequences, as illustrated in FIGS. 51-64.

The VLPs may also be used to produce reagents comprised of recombinantinfluenza structural proteins that self-assemble into functional andimmunogenic homotypic macromolecular protein structures, includingsubviral influenza particles and influenza VLP, in transformed hostscells, for example plant cells or insect cells.

Therefore, the invention provides for VLPs, and a method for producingviral VLPs in a plant expression system, from the expression of a singleenvelope protein. The VLPs may be influenza VLPs, or VLPs produced fromother plasma membrane-derived virus including, but not limited to,Measles, Ebola, Marburg, and HIV.

Proteins from other enveloped viruses, for example but not limited toFiloviridae (e.g. Ebola virus, Marburg virus, or the like),Paramyxoviridae (e.g. Measles virus, Mumps virus, Respiratory syncytialvirus, pneumoviruses, or the like), Retroviridae (e.g. HumanImmunodeficiency Virus-1, Human Immunodeficiency Virus-2, Human T-CellLeukemia Virus-1, or the like), Flaviviridae (e.g. West NileEncephalitis, Dengue virus, Hepatitis C virus, yellow fever virus, orthe like), Bunyaviridae (e.g. Hantavirus or the like), Coronaviridae(e.g. coronavirus, SARS, or the like), as would be known to those ofskill in the art, may also be used. Non limiting examples of antigensthat may be expressed in plasma membrane derived viruses include, thecapsid protein of HIV-p24; HIV glycoproteins gp120 or gp41, Filovirusproteins including VP30 or VP35 of Ebolavirus or Gp/SGP of Marburg virusor the H protein or F protein of the Measles paramyxovirus. For example,P24 of HIV (e.g. GenBank reference gi:19172948) is the protein obtainedby translation and cleavage of the gag sequence of the HIV virus genome(e.g. GenBank reference gi:9629357); gp120 and gp41 of HIV areglycoproteins obtained by translation and cleavage of the gpl 60 protein(e.g. GenBank reference gi:9629363), encoded by env of the HIV virusgenome. VP30 of Ebolavirus (GenPept Reference gi: 55770813) is theprotein obtained by translation of the vp30 sequence of the Ebolavirusgenome (e.g. GenBank Reference gi:55770807); VP35 of Ebolavirus (GenPeptReference gi:55770809) is the protein obtained by translation of thevp35 sequence of the Ebolavirus genome. Gp/SGP of Marburg virus (GenPeptReference gi:296965) is the protein obtained by translation of the(sequence) of the Marburg virus genome (GenBank Reference gi:158539108).H protein (GenPept Reference gi: 9626951) is the protein of the Hsequence of the Measles virus genome (GenBank Reference gi: 9626945); Fprotein (GenPept reference gi: 9626950) is the protein of the F sequenceof the Measles virus genome.

However, other envelope proteins may be used within the methods of thepresent invention as would be know to one of skill in the art.

The invention, therefore, provides for a nucleic acid moleculecomprising a sequence encoding HIV-p24, HIV-gp120, HIV-gp41,Ebolavirus-VP30, Ebolavirus-VP35, Marburg virus Gp/SGP, Measles virus-Hprotein or -F protein. The nucleic acid molecule may be operativelylinked to a regulatory region active in an insect, yeast or plant cell,or in a particular plant tissue.

The present invention further provides the cloning of a nucleic acidencoding an HA, for example but not limited to, human influenzaA/Indonesia/5/05 virus HA (H5N1) into a plant or insect expressionvector (e.g. baculovirus expression vector) and production of influenzavaccine candidates or reagents comprised of recombinant influenzastructural proteins that self-assemble into functional and immunogenichomotypic macromolecular protein structures, including subviralinfluenza particles and influenza VLP, in transformed plant cells ortransformed insect cells.

The nucleic acid encoding the HA of influenza subtypes, for example butnot limited to, A/New Caledonia/20/99 (H1N1), A/Indonesia/5/05 sub-type(H5N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands/3/2006 (H1N1),A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004(H5N1), A/Teal/Hong Kong/W312/97 (H6N1), A/Hong Kong/1073/99 (H9N2),A/Brisbane/10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),A/Equine/Prague/56 (H7N7), B/Malaysia/2506/2004, B/Florida/4/2006 may beexpressed, for example, using a Baculovirus Expression System in anappropriate cell line, for example, Spodoptera frugiperda cells (e.g.Sf-9 cell line; ATCC PTA-4047). Other insect cell lines may also beused.

The nucleic acid encoding the HA may, alternately, be expressed in aplant cell, or in a plant. The nucleic acid encoding HA may besynthesized by reverse transcription and polymerase chain reaction (PCR)using HA RNA. As an example, the RNA may be isolated from humaninfluenza A/New Caledonia/20/99 (H1N1) virus or human influenzaA/Indonesia/5/05 (H5N1) virus, or other influenza viruses e.g.A/Brisbane/59/2007 (H1N1), A/Solomon Islands/3/2006 (H1N1),A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004(H5N1), A/Teal/Hong Kong/W312/97 (H6N1), A/Hong Kong/1073/99 (H9N2),A/Brisbane/10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),A/Equine/Prague/56 (H7N7), B/Malaysia/2506/2004, B/Florida/4/2006, orfrom cells infected with an influenza virus. For reverse transcriptionand PCR, oligonucleotide primers specific for HA RNA, for example butnot limited to, human influenza A/New Caledonia/20/99 (H1N1) virus HAsequences or human influenza A/Indonesia/5/05 (H5N1) virus HA0sequences, or HA sequences from influenza subtypes A/Brisbane/59/2007(H1N1), A/Solomon Islands/3/2006 (H1N1), A/Singapore/1/57 (H2N2),A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1), A/Teal/HongKong/W312/97 (H6N1), A/Hong Kong/1073/99 (H9N2), A/Brisbane/10/2007(H3N2), A/Wisconsin/67/2005 (H3N2), A/Equine/Prague/56 (H7N7),B/Malaysia/2506/2004, B/Florida/4/2006 may be used. Additionally, anucleic acid encoding HA may be chemically synthesized using methods aswould known to one of skill in the art.

The resulting cDNA copies of these genes may be cloned in a suitableexpression vector as required by the host expression system. Examples ofappropriate expression vectors for plants are described below,alternatively, baculovirus expression vector, for example, pFastBacl(InVitrogen), resulting in pFastBacl-based plasmids, using knownmethods, and information provided by the manufacturer's instructions maybe used.

The present invention is further directed to a gene construct comprisinga nucleic acid encoding HA, as described above, operatively linked to aregulatory element that is operative in a plant. Examples of regulatoryelements operative in a plant cell and that may be used in accordancewith the present invention include but are not limited to a plastocyaninregulatory region (U.S. Pat. No. 7,125,978; which is incorporated hereinby reference), or a regulatory region of Ribulose 1,5-bisphosphatecarboxylase/oxygenase (RuBisCO; U.S. Pat. No. 4,962,028; which isincorporated herein by reference), chlorophyll a/b binding protein (CAB;Leutwiler et al; 1986; which is incorporated herein by reference),ST-LS1 (associated with the oxygen-evolving complex of photosystem IIand described by Stockhaus et al. 1987, 1989; which is incorporatedherein by reference). An example of a plastocyanin regulatory region isa sequence comprising nucleotides 10-85 of SEQ ID NO: 36, or a similarregion of any one of SEQ ID NOS: 37-47. A regulatory element orregulatory region may enhance translation of a nucleotide sequence towhich is it operatively linked—the nucleotide sequence may encode aprotein or polypeptide. Another example of a regulatory region is thatderived from the untranslated regions of the Cowpea Mosaic Virus (CPMV),which may be used to preferentially translate the nucleotide sequence towhich it is operatively linked. This CPMV regulatory region comprises aCMPV-HT system—see, for example, Sainsbury et al, 2008, Plant Physiology148: 1212-1218.

If the construct is expressed in an insect cell, examples of regulatoryelements operative in an insect cell include but are not limited to thepolyhedrin promoter (Possee and Howard 1987. Nucleic Acids Research15:10233-10248), the gp64 promoter (Kogan et al, 1995. J Virology69:1452-1461) and the like.

Therefore, an aspect of the invention provides for a nucleic acidcomprising a regulatory region and a sequence encoding an influenza HA.The regulatory region may be a plastocyanin regulatory element, and theinfluenza HA may be selected from a group of influenza strains orsubtypes, comprising A/New Caledonia/20/99 (H1N1), A/Indonesia/5/05sub-type (H5N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands/3/2006(H1N1), A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N1),A/Vietnam/1194/2004 (H5N1), A/Teal/Hong Kong/W312/97 (H6N1), A/HongKong/1073/99 (H9N2), A/Brisbane/10/2007 (H3N2), A/Wisconsin/67/2005(H3N2), A/Equine/Prague/56 (H7N7), B/Malaysia/2506/2004,B/Florida/4/2006. Nucleic acid sequences comprising a plastocyaninregulatory element and an influenza HA are exemplified herein by SEQ IDNOs: 36-47.

It is known that there may be sequence differences in the sequence ofinfluenza hemagglutinin amino acids sequences, or the nucleic acidsencoding them, when influenza virus is cultured in eggs, or mammaliancells, (e.g. MDCK cells) or when isolated from an infected subject.Non-limiting examples of such differences are illustrated herein,including Example 18. Furthermore, as one of skill in the art wouldrealize, additional variation may be observed within influenzahemagglutinins obtained from new strains as additional mutationscontinue to occur. Due to the known sequence variability betweendifferent influenza hemagglutinins, the present invention includes VLPsthat may be made using any influenza hemagglutin provided that whenexpressed in a host as described herein, the influenza hemagglutin formsa VLP.

Sequence alignments and consensus sequences may be determined using anyof several software packages known in the art, for example MULTALIN (F.CORPET, 1988, Nucl. Acids Res., 16 (22), 10881-10890), or sequences maybe aligned manually and similarities and differences between thesequences determined.

The structure of hemagglutinins is well-studied and the structures areknown to be highly conserved. When hemagglutinin structures aresuperimposed, a high degree of structural conservation is observed (rmsd<2A). This structural conservation is observed even though the aminoacid sequence may vary in some positions (see, for example, Skehel andWiley, 2000 Ann Rev Biochem 69:531-69; Vaccaro et al 2005). Regions ofhemagglutinins are also well-conserved, for example:

-   -   Structural domains: The HA0 polyprotein is cleaved to provide        mature HA. HA is a homotrimer with each monomer comprising a        receptor binding domain (HA1) and a membrane-anchoring domain        (HA2) linked by a single disulphide bond; the N-terminal 20        residues of the HA2 subunit may also be referred to as the HA        fusion domain or sequence. A ‘tail’ region (internal to the        membrane envelope) is also present. Each hemagglutinin comprises        these regions or domains. Individual regions or domains are        typically conserved in length.    -   All hemagglutinins contain the same number and position of        intra- and inter-molecular disulfide bridges. The quantity and        position on the amino acid sequence of the cysteines that        participate in disulfide bridge network is conserved among the        HAs. Examples of structures illustrating the characteristic        intra- and intermolecular disulfide bridges and other conserved        amino acids and their relative positions are described in, for        example, Gamblin et al 2004 (Science 303:1838-1842). Exemplary        structures and sequences include 1 RVZ, 1 RVX, 1 RVT, 1 RVO, 1        RUY, 1 RU7, available from the Protein Data Bank (Berman et        al. 2003. Nature Structural Biology 10:980; URL: rcsb.org)    -   Cytoplasmic tail—the majority of hemagglutinins comprise 3        cysteines at conserved positions. One or more of these cysteines        may be palmitoylated as a post-translational modification.

Amino acid variation is tolerated in hemagglutinins of influenzaviruses. This variation provides for new strains that are continuallyidentified. Infectivity between the new strains may vary. However,formation of hemagglutinin trimers, which subsequently form VLPs ismaintained. The present invention, therefore, provides for ahemagglutinin amino acid sequence, or a nucleic acid encoding ahemagglutinin amino acid sequence, that forms VLPs in a plant, andincludes known sequences and variant sequences that may develop.

FIG. 65 illustrates an example of such known variation. This figureshows a consensus amino acid sequence (SEQ ID NO: 74) for HA of thefollowing H1N1 strains:

A/New Caledonia/20/99 (H1N1) (encoded by SEQ ID NO: 33),

A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 48),

A/Solomon Islands/3/2006 (H1N1) (SEQ ID NO: 49) and

SEQ ID NO: 9. X1 (position 3) is A or V; X2 (position 52) is D or N; X3(position 90) is K or R; X4 (position 99) is K or T; X5 (position 111)is Y or H; X6 (position 145) is V or T; X7 (position 154) is E or K; X8(position 161) is R or K; X9 (position 181) is V or A; X10 (position203) is D or N; X11 (position 205) is R or K; X12 (position 210) is T orK; X13 (position 225) is R or K; X14 (position 268) is W or R; X15(position 283) is T or N; X16 (position 290) is E or K; X17 (position432) is I or L; X18 (position 489) is N or D.

As another example of such variation, a sequence alignment and consensussequence for HA of A/New Caledonia/20/99 (H1N1) (encoded by SEQ ID NO:33), A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 48), A/Solomon Islands/3/2006(H1N1) (SEQ ID NO: 49), A/PuertoRico/8/34 (H1N1) and SEQ ID NO: 9 isshown below in Table 3.

TABLE 3 Sequence alignment and consensus sequence for HA of selectedH1N1 strains SEQ ID NO. Sequence1                                                   50 75MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 9MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 48MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 49MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 76.......... .......... .......... .......... .......... Consensusmkxkllvllc tftatyadti cigyhannst dtvdtvlekn vtvthsvnll51                                                 100 75EDSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP 9EDSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP 48ENSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP 49EDSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP 76.......... .......... .......... .......... .......... Consensusexshngklcl lkgiaplqlg ncsvagwilg npecellis. eswsyive.p101                                                150 75NPENGTCYPG YFADYEELRE QLSSVSSFER FEIFPKESSW PNHTVTGVSA 9NPENGTCYPG YFADYEELRE QLSSVSSFER FEIFPKESSW PNHTVTGVSA 48NPENGTCYPG HFADYEELRE QLSSVSSFER FEIFPKESSW PNHTVTGVSA 49NPENGTCYPG HFADYEELRE QLSSVSSFER FEIFPKESSW PNHTTTGVSA 76.......... .......... .......... .......... .......... Consensusnpengtcypg xfadyeelre qlssyssfer feifpkessw pnhtxtgvsa151                                                200 75SCSHNGKSSF YRNLLWLTGK NGLYPNLSKS YVNNKEKEVL VLWGVHHPPN 9SCSHNGKSSF YRNLLWLTGK NGLYPNLSKS YVNNKEKEVL VLWGVHHPPN 48SCSHNGESSF YRNLLWLTGK NGLYPNLSKS YANNKEKEVL VLWGVHHPPN 49SCSHNGESSF YKNLLWLTGK NGLYPNLSKS YANNKEKEVL VLWGVHHPPN 76.......... .......... .......... .......... .......... Consensusscshngxssf yxnllwltgk nglypnlsks yxnnkekevl vlwgvhhppn201                                                250 75IGNQRALYHT ENAYVSVVSS HYSRRFTPEI AKRPKVRDQE GRINYYWTLL 9IGNQRALYHT ENAYVSVVSS HYSRRFTPEI AKRPKVRDQE GRINYYWTLL 48IGDQKALYHT ENAYVSVVSS HYSRKFTPEI AKRPKVRDQE GRINYYWTLL 49IGDQRALYHK ENAYVSVVSS HYSRKFTPEI AKRPKVRDQE GRINYYWTLL 76.......... .....MSLLT EVETYVLSII PSGPLKAEIA QRLEDVFAGK Consensusigxqxalyhx enayvsvvss hysrxftpeI akrPkvr#qe gRi#yywtll251                                                300 75EPGDTIIFEA NGNLIAPWYA FALSRGFGSG IITSNAPMDE CDAKCQTPQG 9EPGDTIIFEA NGNLIAPWYA FALSRGFGSG IITSNAPMDE CDAKCQTPQG 48EPGDTIIFEA NGNLIAPRYA FALSRGFGSG IINSNAPMDK CDAKCQTPQG 49EPGDTIIFEA NGNLIAPRYA FALSRGFGSG IINSNAPMDE CDAKCQTPQG 76NTDLEVLMEW ...LKTRPIL SPLTKGILGF VFTLTVPSER GLQRRRFVQN Consensus#pgdt!ifEa ngnLiapxya faLsrGfgsg !itsnaPm#x cdakcqtpQg301                                                350 75AINSSLPFQN VHPVTIGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI 9AINSSLPFQN VHPVTIGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI 48AINSSLPFQN VHPVTIGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI 49AINSSLPFQN VHPVTIGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI 76ALNG.....N GDPNNMDKAV KLYRKLKREI TFHGAKEISL SYSAGALASC ConsensusAiNsslpfqN vhPvtigecp KyvRsaKlrm vtxGlr#Ips iqSrGlfgai351                                                400 75AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI 9AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI 48AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI 49AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI 76MGLIYNRM.G AVTTEVAFGL VCATCEQIAD SQHRSHRQMV TTTNPLIRHE ConsensusaGfIeggwtG mVdgwyg%hh qneqgsgyAa dQkstqnain giTNkvnsvi401                                                450 75EKMNTQFTAV GKEFNKLERR MENLNKKVDD GFLDIWTYNA ELLVLLENER 9EKMNTQFTAV GKEFNKLERR MENLNKKVDD GFLDIWTYNA ELLVLLENER 48EKMNTQFTAV GKEFNKLERR MENLNKKVDD GFIDIWTYNA ELLVLLENER 49EKMNTQFTAV GKEFNKLERR MENLNKKVDD GFIDIWTYNA ELLVLLENER 76NRMVLASTTA .KAMEQMAGS SEQAAEAMEV A........S QARQMVQAMR Consensus#kMntqfTav gKef#k$err mE#lnkkv#d gfxdiwtyna #llv$l#neR451                                                500 75TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG CFEFYHKCNN ECMESVKNGT 9TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG CFEFYHKCNN ECMESVKNGT 48TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG CFEFYHKCND ECMESVKNGT 49TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG CFEFYHKCND ECMESVKNGT 76TIGTHPSSSA GLKNDLLENL QAYQKRMGVQ MQRFK..... .......... ConsensusTldfHdSnvk nLy#kvks#L knnaKeiGng cfeFyhkcnx ecmesvkngt501                                                550 75YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS LVLLVSLGAI 9YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS LVLLVSLGAI 48YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS LVLLVSLGAI 49YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS LVLLVSLGAI 76.......... .......... .......... .......... .......... Consensusydypkysees klnrekidgv klesmgvyqi laiystvass lvllvslgai 551           56675 SFWMCSNGSL QCRICI 9 SFWMCSNGSL QCRICI 48 SFWMCSNGSL QCRICI 49SFWMCSNGSL QCRICI 76 .......... ...... Consensus sfwmcsngsl qcriciThe consensus sequence indicates in upper case letters amino acidscommon to all sequences at a designated position; lower case lettersindicate amino acids common to at least half, or a majority of thesequences; the symbol ! is any one of I or V; the symbol $ is any one ofL or M; the symbol % is any one of F or Y; the symbol # is any one of N,D, Q, E, B or Z; the symbol “.” is no amino acid (e.g. a deletion); X atposition 3 is any one of A or V; X at position 52 is any one of E or N;X at position 90 is K or R; X at position 99 is T or K; X at position111 is any one of Y or H; X at position 145 is any one of V or T; X atposition 157 is K or E; X at position 162 is R or K; X at position 182is V or A; X at position 203 is N or D; X at position 205 is R or K; Xat position 210 is T or K; X at position 225 is K or Y; X at position333 is H or a deletion; X at position 433 is I or L; X at position 49)is N or D.

As another example of such variation, a sequence alignment and consensussequence for HA of A/Anhui/1/2005 (H5N1) (SEQ ID NO: 55),A/Vietnam/1194/2004 (H5N1) and A/Indonesia/5/2006 (H5N1) (SEQ ID NO: 10)is shown below in Table 4.

TABLE 4 Sequence alignment and consensus sequence for HA of selectedH1N1 strains SEQ ID NO. Sequence1                                                   50 10MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILE 56MEKIVLLFAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILE 55MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILE ConsensusMEKIVLLlAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILE51                                                 100 10KTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKAN 56KTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKAN 55KTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKAN ConsensusKTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKAN101                                                150 10PTNDLCYPGS FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSA 56PVNDLCYPGD FNDYEELKHL LSRINHFEKI QIIPKSSWSS HEASLGVSSA 55PANDLCYPGN FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSA ConsensusPxNDLCYPGx FNDYEELKHL LSRINHFEKI QIIPKSSWSd HEASsGVSSA151                                                200 10CPYLGSPSFF RNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDA 56CPYQGKSSFF RNVVWLIKKN STYPTIKRSY NNTNQEDLLV LWGIHHPNDA 55CPYQGTPSFF RNVVWLIKKN NTYPTIKRSY NNTNQEDLLI LWGIHHSNDA ConsensusCPYqGxpSFF RNVVWLIKKN sTYPTIKrSY NNTNQEDLL! LWGIHHpNDA201                                                250 10AEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSG RMEFFWTILK 56AEQTKLYQNP TTYISVGTST LNQRLVPRIA TRSKVNGQSG RMEFFWTILK 55AEQTKLYQNP TTYISVGTST LNQRLVPKIA TRSKVNGQSG RMDFFWTILK ConsensusAEQTkLYQNP TTYIS!GTST LNQRLVPkIA TRSKVNGQSG RM#FFWTILK251                                                300 10PNDAINFESN GNFIAPEYAY KIVKKGDSAI MKSELEYGNC NTKCQTPMGA 56PNDAINFESN GNFIAPEYAY KIVKKGDSTI MKSELEYGNC NTKCQTPMGA 55PNDAINFESN GNFIAPEYAY KIVKKGDSAI VKSEVEYGNC NTKCQTPIGA ConsensusPNDAINFESN GNFIAPEYAY KIVKKGDSaI mKSElEYGNC NTKCQTPmGA301                                                350 10INSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLRNSPQRE SRRKKRGLFG 56INSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLRNSPQRE RRRKKRGLFG 55INSSMPFHNI HPLTIGECPK YVKSNKLVLA TGLRNSPLRE RRRK.RGLFG ConsensusINSSMPFHNI HPLTIGECPK YVKSNrLVLA TGLRNSPqRE rRRKkRGLFG351                                                400 10AIAGFIEGGW QGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNS 56AIAGFIEGGW QGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNS 55AIAGFIEGGW QGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNS ConsensusAIAGFIEGGW QGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNS401                                                450 10IIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTY NAELLVLMEN 56IIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTY NAELLVLMEN 55IIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTY NAELLVLMEN ConsensusIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTY NAELLVLMEN451                                                500 10ERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYHKC DNECMESIRN 56ERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYHKC DNECMESVRN 55ERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYHKC DNECMESVRN ConsensusERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYHKC DNECMES!RN501                                                550 10GTYNYPQYSE EARLKREEIS GVKLESIGTY QILSIYSTVA SSLALAIMMA 56GTYDYPQYSE EARLKREEIS GVKLESIGIY QILSIYSTVA SSLALAIMVA 55GTYDYPQYSE EARLKREEIS GVKLESIGTY QILSIYSTVA SSLALAIMVA ConsensusGTY#YPQYSE EARLKREEIS GVKLESIGtY QILSIYSTVA SSLALAIMvA551             568 10 GLSLWMCSNG SLQCRICI 56 GLSLWMCSNG SLQCRICI 55GLSLWMCSNG SLQCRICI Consensus GLSLWMCSNG SLQCRICIThe consensus sequence indicates in upper case letters amino acidscommon to all sequences at a designated position; lower case lettersindicate amino acids common to at least half, or a majority of thesequences; the symbol ! is any one of I or V; the symbol $ is any one ofL or M; the symbol % is any one of F or Y; the symbol # is any one of N,D, Q, E, B or Z; X at position 102 is any of T, V or A; X t position 110is any of S, D or N; X at position 156 is any of S, K or T.

The above-illustrated and described alignments and consensus sequencesare non-limiting examples of variants in hemagglutinin amino acidsequences that may be used in various embodiments of the invention forthe production of VLPs in a plant.

A nucleic acid encoding an amino acid sequence may be easily determined,as the codons for each amino acid are known in the art. Provision of anamino acid sequence, therefore, teaches the degenerate nucleic acidsequences that encode it. The present invention, therefore, provides fora nucleic acid sequence encoding the hemagglutinin of those influenzastrains and subtypes disclosed herein (e.g. A/New Caledonia/20/99(H1N1)A/Indonesia/5/2006 (H5N1), A/chicken/New York/1995, A/herringgull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00,A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02,A/Turkey/Ontario/6118/68(H8N4), A/shoveler/Iran/G54/03,A/chicken/Germany/V/1949(H10N7), A/duck/England/56(H11N6),A/duck/Alberta/60/76(H12N5), A/Gull/Maryland/704/77(H13N6),A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H15N8), A/black-headedgull/Sweden/5/99(H16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34(H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1),A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),B/Malaysia/2506/2004, B/Florida/4/2006, A/Singapore/1/57 (H2N2),A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1),A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H7N7),A/HongKong/1073/99 (H9N2)), as well as the degerenate sequences thatencode the above hemagglutinins.

Further, an amino acid sequence encoded by a nucleic acid may be easilydetermined, as the codon or codons for each amino acid are known.Provision of a nucleic acid, therefore, teaches an amino acid sequenceencoded by it. The invention, therefore, provides for amino acidsequences of the hemagglutinin of those influenza strains and subtypesdisclosed herein those disclosed herein (e.g. A/New Caledonia/20/99(H1N1)A/Indonesia/5/2006 (H5N1), A/chicken/New York/1995, A/herringgull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00,A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02,A/Turkey/Ontario/6118/68(H8N4), A/shoveler/Iran/G54/03,A/chicken/Germany/V/1949(H10N7), A/duck/England/56(H11N6),A/duck/Alberta/60/76(H12N5), A/Gull/Maryland/704/77(H13N6),A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H15N8), A/black-headedgull/Sweden/5/99(H16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34(H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1),A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),B/Malaysia/2506/2004, B/Florida/4/2006, A/Singapore/1/57 (H2N2),A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1),A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H17N7),A/HongKong/1073/99 (H9N2)).

In plants, influenza VLPs bud from the plasma membrane (see Example 5,and FIG. 19) therefore the lipid composition of the VLPs reflects theirorigin. The VLPs produced according to the present invention comprise HAof one or more than one type or subtype of influenza, complexed withplant derived lipids. Plant lipids can stimulate specific immune cellsand enhance the immune response induced. Plant membranes are made oflipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), andalso contain glycosphingolipids, saponins, and phytosterols.Additionally, lipid rafts are also found in plant plasma membranes—thesemicrodomains are enriched in sphingolipids and sterols. In plants, avariety of phytosterols are known to occur, including stigmasterol,sitosterol, 24-methylcholesterol and cholesterol (Mongrand et al.,2004).

PC and PE, as well as glycosphingolipids can bind to CD1 moleculesexpressed by mammalian immune cells such as antigen-presenting cells(APCs) like dendritic cells and macrophages and other cells including Band T lymphocytes in the thymus and liver (Tsuji M., 2006). CD1molecules are structurally similar to major histocompatibility complex(MHC) molecules of class I and their role is to present glycolipidantigens to NKT cells (Natural Killer T cells). Upon activation, NKTcells activate innate immune cells such as NK cells and dendritic cellsand also activate adaptive immune cells like the antibody-producing Bcells and T-cells.

A variety of phytosterols may be found in a plasma membrane—the specificcomplement may vary depending on the species, growth conditions,nutrient resources or pathogen state, to name a few factors. Generally,beta-sitosterol is the most abundant phytosterol.

The phytosterols present in an influenza VLP complexed with a lipidbilayer, such as an plasma-membrane derived envelope may provide for anadvantageous vaccine composition. Without wishing to be bound by theory,plant-made VLPs complexed with a lipid bilayer, such as aplasma-membrane derived envelope, may induce a stronger immune reactionthan VLPs made in other expression systems, and may be similar to theimmune reaction induced by live or attenuated whole virus vaccines.

Therefore, in some embodiments, the invention provides for a VLPcomplexed with a plant-derived lipid bilayer. In some embodiments theplant-derived lipid bilayer may comprise the envelope of the VLP.

The VLP produced within a plant may include an HA comprisingplant-specific N-glycans. Therefore, this invention also provides for aVLP comprising HA having plant specific N-glycans.

Furthermore, modification of N-glycan in plants is known (see forexample U.S. 60/944,344; which is incorporated herein by reference) andHA having modified N-glycans may be produced. HA comprising a modifiedglycosylation pattern, for example with reduced fucosylated,xylosylated, or both, fucosylated and xylosylated, N-glycans may beobtained, or HA having a modified glycosylation pattern may be obtained,wherein the protein lacks fucosylation, xylosylation, or both, andcomprises increased galatosylation. Furthermore, modulation ofpost-translational modifications, for example, the addition of terminalgalactose may result in a reduction of fucosylation and xylosylation ofthe expressed HA when compared to a wild-type plant expressing HA.

For example, which is not to be considered limiting, the synthesis of HAhaving a modified glycosylation pattern may be achieved by co-expressingthe protein of interest along with a nucleotide sequence encodingbeta-1.4galactosyltransferase (GalT), for example, but not limited tomammalian GalT, or human GalT however GalT from another sources may alsobe used. The catalytic domain of GalT may also be fused to a CTS domain(i.e. the cytoplasmic tail, transmembrane domain, stem region) ofN-acetylglucosaminyl transferase (GNT1), to produce a GNT1-GalT hybridenzyme, and the hybrid enzyme may be co-expressed with HA. The HA mayalso be co-expressed along with a nucleotide sequence encodingN-acetylglucosaminyltrasnferase III (GnT-III), for example but notlimited to mammalian GnT-III or human GnT-III, GnT-III from othersources may also be used. Additionally, a GNT1-GnT-III hybrid enzyme,comprising the CTS of GNT1 fused to GnT-III may also be used.

Therefore the present invention also includes VLP's comprising HA havingmodified N-glycans.

Without wishing to be bound by theory, the presence of plant N-glycanson HA may stimulate the immune response by promoting the binding of HAby antigen presenting cells. Stimulation of the immune response usingplant N glycan has been proposed by Saint-Jore-Dupas et al. (2007).Furthermore, the conformation of the VLP may be advantageous for thepresentation of the antigen, and enhance the adjuvant effect of VLP whencomplexed with a plant derived lipid layer.

By “regulatory region”, “regulatory element” or “promoter” it is meant aportion of nucleic acid typically, but not always, upstream of theprotein coding region of a gene, which may be comprised of either DNA orRNA, or both DNA and RNA. When a regulatory region is active, and inoperative association, or operatively linked, with a gene of interest,this may result in expression of the gene of interest. A regulatoryelement may be capable of mediating organ specificity, or controllingdevelopmental or temporal gene activation. A “regulatory region”includes promoter elements, core promoter elements exhibiting a basalpromoter activity, elements that are inducible in response to anexternal stimulus, elements that mediate promoter activity such asnegative regulatory elements or transcriptional enhancers. “Regulatoryregion”, as used herein, also includes elements that are activefollowing transcription, for example, regulatory elements that modulategene expression such as translational and transcriptional enhancers,translational and transcriptional repressors, upstream activatingsequences, and mRNA instability determinants. Several of these latterelements may be located proximal to the coding region.

In the context of this disclosure, the term “regulatory element” or“regulatory region” typically refers to a sequence of DNA, usually, butnot always, upstream (5′) to the coding sequence of a structural gene,which controls the expression of the coding region by providing therecognition for RNA polymerase and/or other factors required fortranscription to start at a particular site. However, it is to beunderstood that other nucleotide sequences, located within introns, or3′ of the sequence may also contribute to the regulation of expressionof a coding region of interest. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.Most, but not all, eukaryotic promoter elements contain a TATA box, aconserved nucleic acid sequence comprised of adenosine and thymidinenucleotide base pairs usually situated approximately 25 base pairsupstream of a transcriptional start site. A promoter element comprises abasal promoter element, responsible for the initiation of transcription,as well as other regulatory elements (as listed above) that modify geneexpression.

There are several types of regulatory regions, including those that aredevelopmentally regulated, inducible or constitutive. A regulatoryregion that is developmentally regulated, or controls the differentialexpression of a gene under its control, is activated within certainorgans or tissues of an organ at specific times during the developmentof that organ or tissue. However, some regulatory regions that aredevelopmentally regulated may preferentially be active within certainorgans or tissues at specific developmental stages, they may also beactive in a developmentally regulated manner, or at a basal level inother organs or tissues within the plant as well. Examples oftissue-specific regulatory regions, for example see-specific aregulatory region, include the napin promoter, and the cruciferinpromoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau etal., 1994, Plant Cell 14: 125-130). An example of a leaf-specificpromoter includes the plastocyanin promoter (FIG. 1b or SEQ ID NO:23);U.S. Pat. No. 7,125,978, which is incorporated herein by reference).

An inducible regulatory region is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the protein factorthat binds specifically to an inducible regulatory region to activatetranscription may be present in an inactive form, which is then directlyor indirectly converted to the active form by the inducer. However, theprotein factor may also be absent. The inducer can be a chemical agentsuch as a protein, metabolite, growth regulator, herbicide or phenoliccompound or a physiological stress imposed directly by heat, cold, salt,or toxic elements or indirectly through the action of a pathogen ordisease agent such as a virus. A plant cell containing an inducibleregulatory region may be exposed to an inducer by externally applyingthe inducer to the cell or plant such as by spraying, watering, heatingor similar methods. Inducible regulatory elements may be derived fromeither plant or non-plant genes (e.g. Gatz, C. and Lenk, I. R. P., 1998,Trends Plant Sci. 3, 352-358; which is incorporated by reference).Examples, of potential inducible promoters include, but not limited to,tetracycline-inducible promoter (Gatz, C., 1997, Ann. Rev. PlantPhysiol. Plant Mol. Biol. 48, 89-108; which is incorporated byreference), steroid inducible promoter (Aoyama, T. and Chua, N. H.,1997, Plant J. 2, 397-404; which is incorporated by reference) andethanol-inducible promoter (Salter, M. G., et al, 1998, Plant Journal16, 127-132; Caddick, M. X., et al, 1998, Nature Biotech. 16, 177-180,which are incorporated by reference) cytokinin inducible IB6 and CKI1genes (Brandstatter, I. and Kieber, J. J., 1998, Plant Cell 10,1009-1019; Kakimoto, T., 1996, Science 274, 982-985; which areincorporated by reference) and the auxin inducible element, DR5(Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which isincorporated by reference).

A constitutive regulatory region directs the expression of a genethroughout the various parts of a plant and continuously throughoutplant development. Examples of known constitutive regulatory elementsinclude promoters associated with the CaMV 35S transcript. (Odell etal., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991,Plant Cell, 3: 1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121), or tms 2 (U.S. Pat. No. 5,428,147, which is incorporatedherein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994,Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejoet al, 1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and thetobacco translational initiation factor 4A gene (Mandel et al, 1995Plant Mol. Biol. 29: 995-1004). The term “constitutive” as used hereindoes not necessarily indicate that a gene under control of theconstitutive regulatory region is expressed at the same level in allcell types, but that the gene is expressed in a wide range of cell typeseven though variation in abundance is often observed. Constitutiveregulatory elements may be coupled with other sequences to furtherenhance the transcription and/or translation of the nucleotide sequenceto which they are operatively linked. For example, the CMPV-HT system(Sainsbury et al, 2008, Plant Physiology 148: 1212-1218) is derived fromthe untranslated regions of the Cowpea mosaic virus (COMV) anddemonstrates enhanced translation of the associated coding sequence.

By “native” it is meant that the nucleic acid or amino acid sequence isnaturally occurring, or “wild type”.

By “operatively linked” it is meant that the particular sequences, forexample a regulatory element and a coding region of interest, interacteither directly or indirectly to carry out an intended function, such asmediation or modulation of gene expression. The interaction ofoperatively linked sequences may, for example, be mediated by proteinsthat interact with the operatively linked sequences.

The one or more than one nucleotide sequence of the present inventionmay be expressed in any suitable plant host that is transformed by thenucleotide sequence, or constructs, or vectors of the present invention.Examples of suitable hosts include, but are not limited to, agriculturalcrops including alfalfa, canola, Brassica spp., maize, Nicotiana spp.,alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley,sunflower, cotton and the like.

The one or more chimeric genetic constructs of the present invention canfurther comprise a 3′ untranslated region. A 3′ untranslated regionrefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by effecting the addition of polyadenylic acidtracks to the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. One or more of thechimeric genetic constructs of the present invention can also includefurther enhancers, either translation or transcription enhancers, as maybe required. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence.

Non-limiting examples of suitable 3′ regions are the 3′ transcribednon-translated regions containing a polyadenylation signal ofAgrobacterium tumor inducing (Ti) plasmid genes, such as the nopalinesynthase (Nos gene) and plant genes such as the soybean storage proteingenes, the small subunit of the ribulose-1,5-bisphosphate carboxylase(ssRUBISCO; U.S. Pat. No. 4,962,028; which is incorporated herein byreference) gene, the promoter used in regulating plastocyanin expression(Pwee and Gray 1993; which is incorporated herein by reference). Anexample of a plastocyanin promoter is described in U.S. Pat. No.7,125,978 (which is incorporated herein by reference).

As described herein, promoters comprising enhancer sequences withdemonstrated efficiency in leaf expression, have been found to beeffective in transient expression. Without wishing to be bound bytheory, attachment of upstream regulatory elements of a photosyntheticgene by attachment to the nuclear matrix may mediate strong expression.For example up to −784 from the translation start site of the peaplastocyanin gene may be used mediate strong reporter gene expression.

To aid in identification of transformed plant cells, the constructs ofthis invention may be further manipulated to include plant selectablemarkers. Useful selectable markers include enzymes that provide forresistance to chemicals such as an antibiotic for example, gentamycin,hygromycin, kanamycin, or herbicides such as phosphinothrycin,glyphosate, chlorosulfuron, and the like. Similarly, enzymes providingfor production of a compound identifiable by colour change such as GUS(beta-glucuronidase), or luminescence, such as luciferase or GFP, may beused.

Also considered part of this invention are transgenic plants, plantcells or seeds containing the chimeric gene construct of the presentinvention. Methods of regenerating whole plants from plant cells arealso known in the art. In general, transformed plant cells are culturedin an appropriate medium, which may contain selective agents such asantibiotics, where selectable markers are used to facilitateidentification of transformed plant cells. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be used toestablish repetitive generations, either from seeds or using vegetativepropagation techniques. Transgenic plants can also be generated withoutusing tissue cultures.

Also considered part of this invention are transgenic plants, trees,yeast, bacteria, fungi, insect and animal cells containing the chimericgene construct comprising a nucleic acid encoding recombinant HA0 forVLP production, in accordance with the present invention.

The regulatory elements of the present invention may also be combinedwith coding region of interest for expression within a range of hostorganisms that are amenable to transformation, or transient expression.Such organisms include, but are not limited to plants, both monocots anddicots, for example but not limited to corn, cereal plants, wheat,barley, oat, Nicotiana spp, Brassica spp, soybean, bean, pea, alfalfa,potato, tomato, ginseng, and Arabidopsis.

Methods for stable transformation, and regeneration of these organismsare established in the art and known to one of skill in the art. Themethod of obtaining transformed and regenerated plants is not criticalto the present invention.

By “transformation” it is meant the stable interspecific transfer ofgenetic information (nucleotide sequence) that is manifestedgenotypically, phenotypically or both. The interspecific transfer ofgenetic information from a chimeric construct to a host may be heritableand the transfer of genetic information considered stable, or thetransfer may be transient and the transfer of genetic information is notinheritable.

By the term “plant matter”, it is meant any material derived from aplant. Plant matter may comprise an entire plant, tissue, cells, or anyfraction thereof. Further, plant matter may comprise intracellular plantcomponents, extracellular plant components, liquid or solid extracts ofplants, or a combination thereof. Further, plant matter may compriseplants, plant cells, tissue, a liquid extract, or a combination thereof,from plant leaves, stems, fruit, roots or a combination thereof. Plantmatter may comprise a plant or portion thereof which has not beensubjected to any processing steps. A portion of a plant may compriseplant matter. However, it is also contemplated that the plant materialmay be subjected to minimal processing steps as defined below, or morerigorous processing, including partial or substantial proteinpurification using techniques commonly known within the art including,but not limited to chromatography, electrophoresis and the like.

By the term “minimal processing” it is meant plant matter, for example,a plant or portion thereof comprising a protein of interest which ispartially purified to yield a plant extract, homogenate, fraction ofplant homogenate or the like (i.e. minimally processed). Partialpurification may comprise, but is not limited to disrupting plantcellular structures thereby creating a composition comprising solubleplant components, and insoluble plant components which may be separatedfor example, but not limited to, by centrifugation, filtration or acombination thereof. In this regard, proteins secreted within theextracellular space of leaf or other tissues could be readily obtainedusing vacuum or centrifugal extraction, or tissues could be extractedunder pressure by passage through rollers or grinding or the like tosqueeze or liberate the protein free from within the extracellularspace. Minimal processing could also involve preparation of crudeextracts of soluble proteins, since these preparations would havenegligible contamination from secondary plant products. Further, minimalprocessing may involve aqueous extraction of soluble protein fromleaves, followed by precipitation with any suitable salt. Other methodsmay include large scale maceration and juice extraction in order topermit the direct use of the extract.

The plant matter, in the form of plant material or tissue may be orallydelivered to a subject. The plant matter may be administered as part ofa dietary supplement, along with other foods, or encapsulated. The plantmatter or tissue may also be concentrated to improve or increasepalatability, or provided along with other materials, ingredients, orpharmaceutical excipients, as required.

Examples of a subject or target organism that the VLPs of the presentinvention may be administered to include, but are not limited to,humans, primates, birds, water fowl, migratory birds, quail, duck,geese, poultry, chicken, swine, sheep, equine, horse, camel, canine,dogs, feline, cats, tiger, leopard, civet, mink, stone marten, ferrets,house pets, livestock, rabbits, mice, rats, guinea pigs or otherrodents, seal, whale and the like. Such target organisms are exemplary,and are not to be considered limiting to the applications and uses ofthe present invention.

It is contemplated that a plant comprising the protein of interest, orexpressing the VLP comprising the protein of interest may beadministered to a subject or target organism, in a variety of waysdepending upon the need and the situation. For example, the protein ofinterest obtained from the plant may be extracted prior to its use ineither a crude, partially purified, or purified form. If the protein isto be purified, then it may be produced in either edible or non-edibleplants. Furthermore, if the protein is orally administered, the planttissue may be harvested and directly feed to the subject, or theharvested tissue may be dried prior to feeding, or an animal may bepermitted to graze on the plant with no prior harvest taking place. Itis also considered within the scope of this invention for the harvestedplant tissues to be provided as a food supplement within animal feed. Ifthe plant tissue is being feed to an animal with little or not furtherprocessing it is preferred that the plant tissue being administered isedible.

Post-transcriptional gene silencing (PTGS) may be involved in limitingexpression of transgenes in plants, and co-expression of a suppressor ofsilencing from the potato virus Y (HcPro) may be used to counteract thespecific degradation of transgene mRNAs (Brigneti et al., 1998).Alternate suppressors of silencing are well known in the art and may beused as described herein (Chiba et al., 2006, Virology 346:7-14; whichis incorporated herein by reference), for example but not limited to,TEV-p1/HC-Pro (Tobacco etch virus-p1/HC-Pro), BYV-p21, p19 of Tomatobushy stunt virus (TBSV p19), capsid protein of Tomato crinkle virus(TCV-CP), 2b of Cucumber mosaic virus; CMV-2b), p25 of Potato virus X(PVX-p25), p11 of Potato virus M (PVM-p11), p11 of Potato virus S(PVS-p11), p16 of Blueberry scorch virus, (BScV-p16), p23 of Citrustristeza virus (CTV-p23), p24 of Grapevine leafroll-associated virus-2,(GLRaV-2 p24), p10 of Grapevine virus A, (GVA-p10), p14 of Grapevinevirus B (GVB-p14), p10 of Heracleum latent virus (HLV-p10), or p16 ofGarlic common latent virus (GCLV-p16). Therefore, a suppressor ofsilencing, for example, but not limited to, HcPro, TEV-p1/HC-Pro,BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16,CTV-p23, GLRaV-2 p24, GBV-p14, HLV-p10, GCLV-p16 or GVA-p10, may beco-expressed along with the nucleic acid sequence encoding the proteinof interest to further ensure high levels of protein production within aplant.

Furthermore, VLPs may be produced that comprise a combination of HAsubtypes. For example, VLPs may comprise one or more than one HA fromthe subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, H16, type B, or a combination thereof. Selection of the combinationof HAs may be determined by the intended use of the vaccine preparedfrom the VLP. For example a vaccine for use in inoculating birds maycomprise any combination of HA subtypes, while VLPs useful forinoculating humans may comprise subtypes one or more than one ofsubtypes H1, H2, H3, H5, H6, H7, H9 or B. However, other HA subtypecombinations may be prepared depending upon the use of the VLP. In orderto produce VLPs comprising combinations of HA subtypes, the desired HAsubtype may be co-expressed within the same cell, for example a plantcell.

Furthermore, VLPs produced as described herein do not compriseneuraminidase (NA). However, NA may be co-expressed with HA should VLPscomprising HA and NA be desired.

Therefore, the present invention further includes a suitable vectorcomprising the chimeric construct suitable for use with either stable ortransient expression systems. The genetic information may be alsoprovided within one or more than one construct. For example, anucleotide sequence encoding a protein of interest may be introduced inone construct, and a second nucleotide sequence encoding a protein thatmodifies glycosylation of the protein of interest may be introducedusing a separate construct. These nucleotide sequences may then beco-expressed within a plant. However, a construct comprising anucleotide sequence encoding both the protein of interest and theprotein that modifies glycosylation profile of the protein of interestmay also be used. In this case the nucleotide sequence would comprise afirst sequence comprising a first nucleic acid sequence encoding theprotein of interest operatively linked to a promoter or regulatoryregion, and a second sequence comprising a second nucleic acid sequenceencoding the protein that modifies the glycosylation profile of theprotein of interest, the second sequence operatively linked to apromoter or regulatory region.

By “co-expressed” it is meant that two, or more than two, nucleotidesequences are expressed at about the same time within the plant, andwithin the same tissue of the plant. However, the nucleotide sequencesneed not be expressed at exactly the same time. Rather, the two or morenucleotide sequences are expressed in a manner such that the encodedproducts have a chance to interact. For example, the protein thatmodifies glycosylation of the protein of interest may be expressedeither before or during the period when the protein of interest isexpressed so that modification of the glycosylation of the protein ofinterest takes place. The two or more than two nucleotide sequences canbe co-expressed using a transient expression system, where the two ormore sequences are introduced within the plant at about the same timeunder conditions that both sequences are expressed. Alternatively, aplatform plant comprising one of the nucleotide sequences, for examplethe sequence encoding the protein that modifies the glycosylationprofile of the protein of interest, may be transformed, eithertransiently or in a stable manner, with an additional sequence encodingthe protein of interest. In this case, the sequence encoding the proteinthat modifies the glycosylation profile of the protein of interest maybe expressed within a desired tissue, during a desired stage ofdevelopment, or its expression may be induced using an induciblepromoter, and the additional sequence encoding the protein of interestmay be expressed under similar conditions and in the same tissue, toensure that the nucleotide sequences are co-expressed.

The constructs of the present invention can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, micro-injection, electroporation, infiltration, and thelike. For reviews of such techniques see for example Weissbach andWeissbach, Methods for Plant Molecular Biology, Academy Press, New YorkVIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology,2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer inPlants. In Plant Metabolism, 2d Ed. DT. Dennis, D H Turpin, D DLefebrve, D B Layzell (eds), Addison-Wesley, Langmans Ltd. London, pp.561-579 (1997). Other methods include direct DNA uptake, the use ofliposomes, electroporation, for example using protoplasts,micro-injection, microprojectiles or whiskers, and vacuum infiltration.See, for example, Bilang, et al. (Gene 100: 247-250 (1991), Scheid etal. (Mol. Gen. Genet. 228: 104-112, 1991), Guerche et al. (Plant Science52: 111-116, 1987), Neuhause et al. (Theor. Appi Genet. 75: 30-36,1987), Klein et al., Nature 327: 70-73 (1987); Howell et al. (Science208: 1265, 1980), Horsch et al. (Science 227: 1229-1231, 1985), DeBlocket al., Plant Physiology 91: 694-701, 1989), Methods for Plant MolecularBiology (Weissbach and Weissbach, eds., Academic Press Inc., 1988),Methods in Plant Molecular Biology (Schuler and Zielinski, eds.,Academic Press Inc., 1989), Liu and Lomonossoff (J. Virol Meth,105:343-348, 2002), U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792;6,403,865; 5,625,136, (all of which are hereby incorporated byreference).

Transient expression methods may be used to express the constructs ofthe present invention (see Liu and Lomonossoff, 2002, Journal ofVirological Methods, 105:343-348; which is incorporated herein byreference). Alternatively, a vacuum-based transient expression method,as described by Kapila et al. 1997 (incorporated herein by reference)may be used. These methods may include, for example, but are not limitedto, a method of Agro-inoculation or Agro-infiltration, however, othertransient methods may also be used as noted above. With eitherAgro-inoculation or Agro-infiltration, a mixture of Agrobacteriacomprising the desired nucleic acid enter the intercellular spaces of atissue, for example the leaves, aerial portion of the plant (includingstem, leaves and flower), other portion of the plant (stem, root,flower), or the whole plant. After crossing the epidermis theAgrobacterium infect and transfer t-DNA copies into the cells. The t-DNAis episomally transcribed and the mRNA translated, leading to theproduction of the protein of interest in infected cells, however, thepassage of t-DNA inside the nucleus is transient.

If the nucleotide sequence of interest encodes a product that isdirectly or indirectly toxic to the plant, then by using the method ofthe present invention, such toxicity may be reduced throughout the plantby selectively expressing the nucleotide sequence of interest within adesired tissue or at a desired stage of plant development. In addition,the limited period of expression resulting from transient expression mayreduce the effect when producing a toxic product in the plant. Aninducible promoter, a tissue-specific promoter, or a cell specificpromoter, may be used to selectively direct expression of the sequenceof interest.

The recombinant HA VLPs of the present invention can be used inconjunction with existing influenza vaccines, to supplement thevaccines, render them more efficacious, and to reduce the administrationdosages necessary. As would be known to a person of skill in the art,the vaccine may be directed against one or more than one influenzavirus. Examples of suitable vaccines include, but are not limited to,those commercially available from Sanofi-Pasteur, ID Biomedical, Merial,Sinovac, Chiron, Roche, Medlmmune, GlaxoSmithKline, Novartis,Sanofi-Aventis, Serono, Shire Pharmaceuticals and the like.

If desired, the VLPs of the present invention may be admixed with asuitable adjuvant as would be known to one of skill in the art.Furthermore, the VLP may be used in a vaccine composition comprising aneffective dose of the VLP for the treatment of a target organism, asdefined above. Furthermore, the VLP produced according to the presentinvention may be combined with VLPs obtained using different influenzaproteins, for example, neuraminidase (NA).

Therefore, the present invention provides a method for inducing immunityto influenza virus infection in an animal or target organism comprisingadministering an effective dose of a vaccine comprising one or more thanone VLP. The vaccine may be administered orally, intradermally,intranasally, intramuscularly, intraperitoneally, intravenously, orsubcutaneously.

Administration of VLPs produced according to the present invention isdescribed in Example 6. Administration of plant-made H5 VLP resulted ina significantly higher response when compared to administration ofsoluble HA (see FIGS. 21A and 21B).

As shown in FIGS. 26A and 26B a subject administered A/Indonesia/5/05 H5VLPs is provided cross-protection to a challenge with influenzaA/Turkey/582/06 (H5N1; “Turkey H5N1”). Administration of Indonesia H5VLPs before challenge did not result in any loss of body mass. Howeverin subject not administered H5 VLPs, but challenged with Turkey H5N1,exhibited significant loss of body mass, and several subject died.

These data, therefore, demonstrate that plant-made influenza VLPscomprising the H5 hemagglutinin viral protein induce an immune responsespecific for pathogenic influenza strains, and that virus-like particlesmay bud from a plant plasma membrane.

Therefore, the present invention provides a composition comprising aneffective dose of a VLP comprising an influenza virus HA protein, one ormore than one plant lipid, and a pharmaceutically acceptable carrier.The influenza virus HA protein may be H5 Indonesia/5/2006,A/Brisbane/50/2007, A/Sololmon Islands 3/2006, A/Brisbane/10/2007,A/Wisconsin/67/2005, B/Malaysia/2506/2005, B/Florida/4/2006,A/Singapore/1/57, A/Anhui/1/2005, A/Vietnam/1194/2004,A/Teal/HongKong/W312/97, A/Equine/Prague/56 or A/HongKong/1073/99. Alsoprovided is a method of inducing immunity to an influenza virusinfection in a subject. The method comprising administering the viruslike particle comprising an influenza virus HA protein, one or more thanone plant lipid, and a pharmaceutically acceptable carrier. The viruslike particle may be administered to a subject orally, intradermally,intranasally, intramusclarly, intraperitoneally, intravenously, orsubcutaneously.

Compositions according to various embodiments of the invention maycomprise VLPs of two or more influenza strains or subtypes. “Two ormore” refers to two, three, four, five, six, seven, eight, nine, 10 ormore strains or subtypes. The strains or subtypes represented may be ofa single subtype (e.g. all H1N1, or all H5N1), or may be a combinationof subtypes. Exemplary subtype and strains include, but are not limitedto, those disclosed herein (e.g. A/New Caledonia/20/99(H1N1)A/Indonesia/5/2006 (H5N1), A/chicken/New York/1995, A/herringgull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00,A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02,A/Turkey/Ontario/6118/68(H8N4), A/shoveler/Iran/G54/03,A/chicken/Germany/N/1949(H10N7), A/duck/England/56(H11N6),A/duck/Alberta/60/76(H12N5), A/Gull/Maryland/704/77(H13N6),A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H15N8), A/black-headedgull/Sweden/5/99(H16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34(H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1),A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2),B/Malaysia/2506/2004, B/Florida/4/2006, A/Singapore/1/57 (H2N2),A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1),A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H7N7),A/HongKong/1073/99 (H9N2)).

The choice of combination of strains and subtypes may depend on thegeographical area of the subjects likely to be exposed to influenza,proximity of animal species to a human population to be immunized (e.g.species of waterfowl, agricultural animals such as swine, etc) and thestrains they carry, are exposed to or are likely to be exposed to,predictions of antigenic drift within subtypes or strains, orcombinations of these factors. Examples of combinations used in pastyears are available (see URL: who.int/csr/dieease/influenza/vaccinerecommendations1/en). Some or all of these strains may be employed inthe combinations shown, or in other combinations, in the production of avaccine composition.

More particularly, exemplary combinations may include VLPs from two ormore strains or subtypes selected from the group comprising:A/Brisbane/59/2007 (H1N1), an A/Brisbane/59/2007 (H1N1)-like virus,A/Brisbane/10/2007 (H3N2), an A/Brisbane/10/2007 (H3N2)-like virus,B/Florida/4/2006 or an B/Florida/4/2006-like virus.

Another exemplary combination may include VLPs from two or more strainsor subtypes selected from the group comprising A/Indonesia/5/2005, anA/Indonesia/5/2005-like virus, A/Vietnam/1194/2004, anA/Vietnam/1194/2004-like virus, A/Anhui/1/05, an A/Anhui/1/05-likevirus, A/goose/Guiyang/337/2006, A/goose/Guiyang/337/2006—like virus,A/chicken/Shanxi/2/2006, or A/chicken/Shanxi/2/2006-like virus.

Another exemplary combination may include VLPs ofA/Chicken/Italy/13474/99 (H7 type) or A/Chicken/British Columbia/04(H7N3) strains of influenza.

Another exemplary combination may include VLPs ofA/Chicken/HongKong/G9/97 or A/HongKong/1073/99. Another exemplarycombination may comprise VLPs of A/Solomon Islands/3/2006. Anotherexemplary combination may comprise VLPs of A/Brisbane/10/2007. Anotherexemplary combination may comprise VLPs of A/Wisconsin/67/2005. Anotherexemplary combination may comprise VLPs of the B/Malaysia/2506/2004,B/Florida/4/2006 or B/Brisbane/3/2007 strains or subtypes.

The two or more VLPs may be expressed individually, and the purified orsemi-purified VLPs subsequently combined. Alternately, the VLPs may beco-expressed in the same host, for example a plant. The VLPs may becombined or produced in a desired ratio, for example about equivalentratios, or may be combined in such a manner that one subtype or straincomprises the majority of the VLPs in the composition.

Therefore, the invention provides for compositions comprising VLPs oftwo or more strains or subtypes.

VLPs of enveloped viruses generally acquire their envelope from themembrane they bud through. Plant plasma membranes have a phytosterolcomplement that may have immunostimulatory effects. To investigate thispossibility, plant-made H5 VLPs were administered to animals in thepresence or absence of an adjuvant, and the HAI (hemagglutinationinhibition antibody response) determined (FIGS. 22A, 22B). In theabsence of an added adjuvant plant-made H5 VLPs demonstrate asignificant HAI, indicative of a systemic immune response toadministration of the antigen. Furthermore, the antibody isotypeprofiles of VLPs administered in the present or absence of adjuvant aresimilar (FIG. 23A).

Table 5 lists sequences provided in various embodiments of theinvention.

TABLE 5 Sequence description for sequence identifiers. SEQ ID NoSequence Description In Disclosure 1 N terminal H1 fragment FIG. 4a 2C terminal H1 fragment FIG. 4b 3 H5 coding sequence FIG. 6 4primer Plato-443c FIG. 7a 5 primer SpHA(Ind)-Plasto.r FIG. 7b 6primer Plasto-SpHA(Ind).c FIG. 7c 7 primer HA(Ind)-Sac.r FIG. 7d 8Sequence of the alfalfa plastocyanin-based FIG. 1expression cassette used for the expression of H1 9HA1 peptide sequence(A/New FIG. 8a Caledonia/20/99) 10HA5 peptide sequence FIG. 8b (A/Indonesia/5/2006) 11Influenza A Subtype H7 coding sequence FIG. 9 (A/chicken/New York/1995)12 Influenza A Subtype H2 coding sequence FIG. 10a(A/herring gull/DE/677/88 (H2N8)) 13Influenza A Subtype H3 coding sequence FIG. 10b (A/Texas/32/2003) 14Influenza A Subtype H4 coding sequence FIG. 10c (A/mallard/MN/33/00) 15Influenza A Subtype H5 coding sequence FIG. 10d (A/duck/Shanghai/1/2000)16 Influenza A Subtype H6 coding sequence FIG. 10e(A/northern pintail/TX/828189/02) 17Influenza A Subtype H8 coding sequence FIG. 10f(A/Turkey/Ontario/6118/68(H8N4)) 18Influenza A Subtype H9 coding sequence FIG. 10g (A/shoveler/Iran/G54/03)19 Influenza A Subtype H10 coding sequence FIG. 10h(A/chicken/Germany/N/1949 (H10N7)) 20Influenza A Subtype H11 coding sequence FIG. 10i(A/duck/England/56(H11N6)) 21 Influenza A Subtype H12 coding sequenceFIG. 10j (A/duck/Alberta/60/76(H12N5)) 22Influenza A Subtype H13 coding sequence FIG. 10k(A/Gull/Maryland/704/77 (H13N6)) 23Influenza A Subtype H14 coding sequence FIG. 10l(A/Mallard/Gurjev/263/82) 24 Influenza A Subtype H15 coding sequenceFIG. 10m (A/duck/Australia/341/83 (H15N8)) 25Influenza A Subtype H16 coding sequence FIG. 10n(A/black-headed gull/Sweden/5/99(H16N3)) 26Influenza B HA coding sequence FIG. 10o (B/Lee/40) 27Influenza C HA coding sequence FIG. 10p (C/Johannesburg/66) 28Complete HAO H1 sequence FIG. 5 29 Primer XmaI-pPlas.c FIG. 10q 30Primer SacI-ATG-pPlas.r FIG. l0r 31 Primer SacI-PlasTer.c FIG. 10s 32Primer EcoRI-PlasTer.r FIG. 10t 33 A/New Caledonia/20/99 (H1N1) FIG. 16GenBank Accession No. AY289929 34 M. Sativa protein disulfide isomeraseFIG. 17 GenBank Accession No. Z11499 35 A/.PuertoRico/8/34 (H1N1)FIG. 18 GenBank Accession No. NC_002016.1 36Clone 774: DNA from DraIII to Sac1 FIG. 28comprising plastocyanin regulatory regionoperatively linked to sequence encoding HA of A/Brisbane/59/2007 (H1N1)37 Clone 775: DNA from DraIII to FIG. 29Sac1comprising plastocyanin regulatoryregion operatively linked to sequenceencoding HA of A/Solomon Islands 3/2006 (H1N1) 38Clone 776: DNA from DraIII to FIG. 30Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of A/Brisbane 10/2007(H3N2) 39 Clone 777: DNA from DraIII to FIG. 31Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of A/Wisconsin/67/2005(H3N2) 40 Clone 778: DNA from DraIII to FIG. 32Sac1comprising plastocyanin regulatoryregion operatively linked to sequenceencoding HA of B/Malaysia/2506/2004 41 Clone 779: DNA from DraIII toFIG. 33 Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of B/Florida/4/2006 42Clone 780: DNA from DraIII to FIG. 34Sac1comprising plastocyanin regulatoryregion operatively linked to sequenceencoding HA of A/Singapore/1/57 (H2N2) 43 Clone 781: DNA from DraIII toFIG. 35 Sac1comprising plastocyanin regulatoryregion operatively linked to sequenceencoding HA of A/Anhui/1/2005 (H5N1) 44 Clone 782: DNA from DraIII toFIG. 36 Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of A/Vietnam/1194/2004(H5N1) 45 Clone 783: DNA from DraIII to FIG. 37Sac1comprising plastocyanin regulatoryregion operatively linked to sequenceencoding HA of A/Teal/HongKong/W312/97 (H6N1) 46Clone 784: DNA from DraIII to FIG. 38Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of A/Equine/Prague/56(H7N7) 47 Clone 785: DNA from DraIII to FIG. 39Sac1comprising plastocyanin regulatoryregion operatively linked to sequence encoding HA of A/HongKong/1073/99(H9N2) 48 Clone 774 HA amino acid sequence FIG. 40AA/Brisbane/59/2007 (H1N1) 49 Clone 775 HA amino acid sequence FIG. 40BA/Solomon Islands 3/2006 (HIN1) 50 Clone 776 HA amino acid sequenceFIG. 41A A/Brisbane 10/2007 (H3N2) 51 Clone 777 HA amino acid sequenceFIG. 41B A/Wisconsin/67/2005 (H3N2) 52 Clone 778 HA amino acid sequenceFIG. 42A B/Malaysia/2506/2004 53 Clone 779 HA amino acid sequenceFIG. 42B B/Florida/4/2006 54 Clone 780 HA amino acid sequence FIG. 43AA/Singapore/1/57 (H2N2) 55 Clone 781 HA amino acid sequence FIG. 43BA/Anhui/1/2005 (H5N1) 56 Clone 782 HA amino acid sequence FIG. 44AA/Vietnam/1194/2004 (H5N1) 57 Clone 783 HA amino acid sequence FIG. 44BA/Teal/HongKong/W312/97 (H6N1) 58 Clone 784 HA amino acid sequenceFIG. 45A A/Equine/Prague/56 (H7N7) 59 Clone 785 HA amino acid sequenceFIG. 45B A/HongKong/1073/99 (H9N2) 60HA expression cassette comprising alfalfa FIG. 51plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 from A/Indonesia/5/2005 (Construct #660), alfalfa plastocyanin 3′ UTR and terminator sequences 61HA expression cassette comprising alfalfa FIG. 52plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 fromA/New Caledonia/20/1999 (Construct # 540), alfalfa plastocyanin 3′UTR and terminator sequences 62HA expression cassette comprising alfalfa FIG. 53plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 fromA/Brisbane/59/2007 (construct #774), alfalfa plastocyanin 3′UTR and terminator sequences 63HA expression cassette comprising alfalfa FIG. 54plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H1 from A/Solomon Islands/3/2006 (H1N1)(construct #775), alfalfa plastocyanin 3′ UTR and terminator sequences64 HA expression cassette comprising alfalfa FIG. 55plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H2 fromA/Singapore/1/57 (H2N2) (construct # 780), alfalfa plastocyanin 3'UTR and terminator sequences 65HA expression cassette comprising alfalfa FIG. 56plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 fromA/Anhui/1/2005 (H5N1) (Construct# 781), alfalfa plastocyanin 3′ UTR andterminator sequences 66 HA expression cassette comprising alfalfaFIG. 57 plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H5 fromA/Vietnam/1194/2004 (H5N1) (Construct # 782), alfalfa plastocyanin 3′UTR and terminator sequences 67HA expression cassette comprising alfalfa FIG. 58plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H6 from A/Teal/Hong Kong/W312/97 (H6N1)(Construct # 783), alfalfa plastocyanin 3′ UTR and terminator sequences68 HA expression cassette comprising alfalfa FIG. 59plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H9 fromA/Hong Kong/1073/99 (H9N2) (Construct # 785), alfalfa plastocyanin 3′UTR and terminator sequences 69HA expression cassette comprising alfalfa FIG. 60plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H3 fromA/Brisbane/10/2007 (H3N2), alfalfa plastocyanin 3′ UTR and terminatorsequences 70 HA expression cassette comprising alfalfa FIG. 61plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H3 fromA/Wisconsin/67/2005 (H3N2), alfalfa plastocyanin 3′ UTR and terminatorsequences 71 HA expression cassette comprising alfalfa FIG. 62plastocyanin promoter and 5′ UTR,hemagglutinin coding sequence of H7 fromA/Equine/Prague/56 (H7N7), alfalfa plastocyanin 3′ UTR and terminatorsequences 72 HA expression cassette comprising alfalfa FIG. 63plastocyanin promoter and 5′ UTR, hemagglutinin coding sequence of HAfrom B/Malaysia/2506/2004, alfalfa plastocyanin 3′ UTR and terminatorsequences 73 HA expression cassette comprising alfalfa FIG. 64plastocyanin promoter and 5′ UTR, hemagglutinin coding sequence of HAfrom B/Florida/4/2006, alfalfa plastocyanin 3′ UTR and terminatorsequences 74 Consensus amino acid sequence of SEQ ID FIG. 65NO: 49, 48, 33 and 9 75 Amino acid sequence of H1 New Caledonia FIG. 66(AAP34324.1) encoded by SEQ ID NO: 33 76Amino acid sequence of H1 Puerto Rico FIG. 67(NC_0409878.1) encoded by SEQ ID NO: 35 77 pBinPlus.2613c AGGAAGGGAAGAAAGCGAAAGGAG 78 Mut-ATG115.r GTGCCGAAGCACGAT CTGACAACGTTGAAG ATCGCT CACGCAAGA AAGACAAGAGA 79 Mut-ATG161.c GTTGTCAGATCGTGC TTCGGCACCAGTACA ACGTTTTCTTTCACTG AAGCGA 80 LC-C5-1.110r TCTCCTGGAGTCACA GACAGGGTGG 81Expression cassette number 828, from PacI FIG. 68(upstream promoter) to AscI (immediately downstream NOS terminator). 82SpPDI-HA(Ind).c GTTCCTTCTCAGATCT TCGCTGATCAGATTT GCATTGGTTACCATG CA 83Construct number 663, from HindIII (in the FIG. 69multiple cloning site, upstream Plastocyanine promoter to EcoRI(immediately downstream Plastocynine terminator). 84 SpPDI-H1B.cTTCTCAGATCTTCG CTGACACAATATGT ATAGGCTACCATGC TAACAAC 85 SacI-H1B.r CTTAGAGCTC TTAG ATGCATATTCTACA CTGTAAAGACCCAT TGGAA 86Construct number 787, from HindIII (in the FIG. 70multiple cloning site, upstream Plastocyanine promoter) to EcoRI(immediately downstream Plastocynine terminator) 87 H3B-SpPDI.rTGTCATTTCCGGGA AGTTTTTG AGCGAA GATCTGAGAAGGA ACCA 88 SpPDI-H3B.cTCTCAGATCTTCG CT CAAAAACTTCCC GGAAATGACAACA GCACG 89 H3 (A-Bri).982rTTGCTTAACATATC TGGGACAGG 90 Construct number 790, from HindIII (in theFIG. 7 multiple cloning site, upstream Plastocyanine promoter) to EcoRI(immediately downstream Plastocynine terminator). 91 HBF-SpPDI.rGTTATTCCAGTGCA GATTCGATCAGCGA AGATCTGAGAAGG AACCAACAC 92 SpPDI-HBF.cCAGATCTTCGCTGA TCGAATCTGCACTG GAATAACATCTTCA AACTCACC 93 Plaster80rCAAATAGTATTTCA TAACAACAACGATT 94Construct number 798, from HindIII (in the FIG. 72multiple cloning site, upstream Plastocyanine promoter) to EcoRI(immediately downstream Plastocynine terminator). 95 ApaI-SpPDI.c TTGTCGGGCCC AT GGCGAAAAACGTT GCGATTTTCGGCTT ATTGT 96 StuI-H1(A-NC).r AAAATAGGCCT TT AGATGCATATTCTA CACTGCAAAGACCC A 97Construct number 580, from PacI (upstream FIG. 7335S promoter) to AscI (immediately downstream NOS terminator). 98ApaI-H5 (A-Indo).1c TGTC GGGCCC ATG GAGAAAATAGTGCT TCTTCTTGCAAT 99H5 (A-Indo)-StuI.1707r AAAT AGGCCT TTA AATGCAAATTCTGC ATTGTAACGA 100Construct number 685, from PacI (upstream FIG. 7435S promoter) to AscI (immediately downstream NOS terminator). 101Construct number 686, from PacI (upstream FIG. 7535S promoter) to AscI (immediately downstream NOS terminator) 102ApaI-H1B.c TGTC GGGCCC ATG AAAGTAAAACTACT GGTCCTGTTATGCA CATT 103StuI-H2B.r AAAT AGGCCT TTA GATGCATATTCTAC ACTGTAAAGACCCA TTGGA 104Construct 732, from PacI (upstream 35S FIG. 76promoter) to AscI (immediately downstream NOS terminator). 105Construct number 733, from PacI (upstream FIG. 7735S promoter) to AscI (immediately downstream NOS terminator). 106ApaI-H3B.c TTGTC GGGCCC AT GAAGACTATCATTG CTTTGAGCTACATT CTATGTC 107StuI-H3B.r AAAAT AGGCCT TC AAATGCAAATGTTG CACCTAATGTTGCC TTT 108Construct number 735, from PacI (upstream FIG. 7835S promoter) to AscI (immediately downstream NOS terminator). 109Construct number 736, from PacI (upstream FIG. 7935S promoter) to AscI (immediately downstream NOS terminator). 110ApI-HBF.c TTGTC GGGCCC AT GAAGGCAATAATTG TACTACTCATGGTA GTAAC 111StuI-HBF.r AAAAT AGGCCT TT ATAGACAGATGGA GCATGAAACGTTGT CTCTGG 112Construct number 738, from PacI (upstream FIG. 8035S promoter) to AscI (immediately downstream NOS terminator). 113Construct number 739, from PacI (upstream FIG. 8135S promoter) to AscI (immediately downstream NOS terminator). 114M. sativa Msj1 coding sequence FIG. 82 115 Hsp-40Luz.1c ATGTTTGGGCGCGGACCAAC 116 Hsp40Luz-SacI.1272r AGCT

CTAC TGTTGAGCGCATTG CAC 117 Hsp40Luz-Plasto.r GTTGGTCCGCGCCCAAACATTTTCTCTC AAGATGAT 118 Hsp70Ara.1c ATGTCGGGTAAAGG AGAAGGA 119Hsp70Ara-SacI.1956r AGCT

TTAG TCGACCTCCTCGAT CTTAG 120 Hsp70Ara-Plasto.r TCCTTCTCCTTTACCCGACATTTTCTCTC AAGATGAT 121 Construct number R850, from HindIII (inFIG. 83 the multiple cloning site, upstreampromoter) to EcoRI (immediately downstream NOS terminator). 122Construct number R860, from HindIII (in FIG. 84the multiple cloning site, upstream promoter) to EcoRI (immediatelydownstream NOS terminator) 123 Construct number R870, from HindIII (inFIG. 85 the multiple cloning site, upstreampromoter) to EcoRI (immediately downstream NOS terminator). 124supP19-plasto.r CCTTGTATAGCTCG TTCCATTTTCTCTCA AGATG 125 supP19-1cATGGAACGAGCTAT ACAAGG 126 SupP19-SacI.r AGTCGAGCTCTTAC TCGCTTTCTTTTTCGAAG The invention will now be described in detail by way of referenceonly to the following non-limiting examples.Methods and Materials1. Assembly of Plastocyanin-Based Expression Cassettes for Native HA

All manipulations were done using the general molecular biologyprotocols of Sambrook and Russell (2001; which is incorporated herein byreference). The first cloning step consisted in assembling a receptorplasmid containing upstream and downstream regulatory elements of thealfalfa plastocyanin gene. The plastocyanin promoter and 5′UTR sequenceswere amplified from alfalfa genomic DNA using oligonucleotide primersXmaI-pPlas.c (SEQ ID NO: 29; FIG. 10Q) and SacI-ATG-pPlas.r (SEQ ID NO:30; FIG. 10R). The resulting amplification product was digested withXmaI and SacI and ligated into pCAMBIA2300 (Cambia, Canberra,Australia), previously digested with the same enzymes, to createpCAMBIApromo Plasto. Similarly, the 3′UTR sequences and terminator ofthe plastocyanin gene was amplified from alfalfa genomic DNA using thefollowing primers: SacI-PlasTer.c (SEQ ID NO: 31; FIG. 10S) andEcoRI-PlasTer.r (SEQ ID NO: 32; FIG. 10T), and the product was digestedwith SacI and EcoRI before being inserted into the same sites ofpCAMBIApromoPlasto to create pCAMBIAPlasto.

A fragment encoding hemagglutinin from influenza strain A/Indonesia/5/05(H5N1; Acc. No. LANL ISDN125873) was synthesized by Epoch Biolabs (SugarLand, Tex., USA). The fragment produced, containing the complete H5coding region including the native signal peptide flanked by a HindIIIsite immediately upstream of the initial ATG, and a SacI siteimmediately downstream of the stop (TAA) codon, is presented in SEQ IDNO: 3 (FIG. 6). The H5 coding region was cloned into aplastocyanin-based expression cassette by the PCR-based ligation methodpresented in Darveau et al. (1995). Briefly, a first PCR amplificationwas obtained using primers Plato-443c (SEQ ID NO: 4; FIG. 7A) andSpHA(Ind)-Plasto.r (SEQ ID NO:5; FIG. 7B) and pCAMBIA promoPlasto astemplate. In parallel, a second amplification was performed with primersPlasto-SpHA(Ind).c (SEQ ID NO: 6; FIG. 7C) and HA(Ind)-Sac.r (SEQ IDNO:7; FIG. 7D) with H5 coding fragment as template. The amplificationobtained from both reactions were mixed together and the mixture servedas template for a third reaction (assembling reaction) using Plato-443c(SEQ ID NO: 4; FIG. 7A) and HA(Ind)-Sacs (SEQ ID NO: 7; FIG. 7D) asprimers. The resulting fragment was digested with BamHI (in theplastocyanin promoter) and SacI (at the 3′ end of the fragment) andcloned into pCAMBIAPlasto previously digested with the same enzymes. Theresulting plasmid, named 660, is presented in FIG. 2B (also see FIG.11).

Hemagglutinin expression cassettes number 774 to 785 were assembled asfollows. A synthetic fragment was synthesized comprising the completehemagglutinin coding sequence (from ATG to stop) flanked in 3′ byalfalfa plastocyanin gene sequences corresponding to the first 84nucleotides upstream of the plastocyanin ATG and ending with a DraIIIrestriction site. The synthetic fragments also comprised a SacI siteimmediately after the stop codon.

Synthetic hemagglutinin fragments were synthesized by Top GeneTechnologies (Montreal, QC, Canada) and Epoch Biolabs (Sugar Land, Tex.,USA). The fragment synthesized are presented in FIGS. 28 to 39 andcorrespond to SEQ ID NO:36 to SEQ ID NO:47. For the assembly of thecomplete expression cassettes, the synthetic fragments were digestedwith DraIII and SacI and cloned into pCAMBIAPlasto previously digestedwith the same enzymes. Table 6 presents the cassettes produced with thecorresponding HA and other references in the text.

TABLE 6 Hemagglutinin expression cassettes assembled from DraIII-SacIsynthetic fragments. Synthetic fragment Complete cassette CassetteSynthetic fragment Final cassette number Corresponding HA Figure SEQ IDNO Figure SEQ ID NO 774 HA of A/Brisbane/59/2007 (H1N1) 28 36 53 62 775HA of A/Solomon Islands 3/2006 (H1N1) 29 37 54 63 776 HA of A/Brisbane10/2007 (H3N2) 30 38 60 69 777 HA of A/Wisconsin/67/2005 (H3N2) 31 39 6170 778 HA of B/Malaysia/2506/2004 32 40 63 72 779 HA of B/Florida/4/200633 41 64 73 780 HA of A/Singapore/1/57 (H2N2) 34 42 55 64 781 HA ofA/Anhui/1/2005 (H5N1) 35 43 56 65 782 HA of A/Vietnam/1194/2004 (H5N1)36 44 57 66 783 HA of A/Teal/HongKong/W312/97 (H6N1) 37 45 58 67 784 HAof A/Equine/Prague/56 (H7N7) 38 46 62 71 785 HA of A/HongKong/1073/99(H9N2) 39 47 59 68Assembly of Plastocyanin-Based PDISP/HA-Fusion Expression CassettesH1 A/New Caledonia/20/99 (Construct Number 540)

The open reading frame from the H1 gene of influenza strain A/NewCaledonia/20/99 (H1N1) was synthesized in two fragments (PlantBiotechnology Institute, National Research Council, Saskatoon, Canada).A first fragment synthesized corresponds to the wild-type H1 codingsequence (GenBank acc. No. AY289929; SEQ ID NO: 33; FIG. 16) lacking thesignal peptide coding sequence at the 5′ end and the transmembranedomain coding sequence at the 3′ end. A BglII restriction site was addedat the 5′ end of the coding sequence and a dual SacI/StuI site was addedimmediately downstream of the stop codon at the 3′ terminal end of thefragment, to yield SEQ ID NO: 1 (FIG. 5A). A second fragment encodingthe C-terminal end of the H1 protein (comprising a transmembrane domainand cytoplasmic tail) from the KpnI site to the stop codon, and flankedin 3′ by SacI and StuI restriction sites was also synthesized (SEQ IDNO. 2; FIG. 5B).

The first H1 fragment was digested with BglII and SacI and cloned intothe same sites of a binary vector (pCAMBIAPlasto) containing theplastocyanin promoter and 5′UTR fused to the signal peptide of alfalfaprotein disulfide isomerase (PDI) gene (nucleotides 32-103; AccessionNo. Z11499; SEQ ID NO: 34; FIG. 17) resulting in a PDI-H1 chimeric genedownstream of the plastocyanin regulatory elements. The sequence of theplastocyanin-based cassette containing the PDI signal peptide ispresented in FIG. 1 (SEQ ID NO:8). The resulting plasmid contained H1coding region fused to the PDI signal peptide and flanked byplastocyanin regulatory elements. The addition of the C-terminal endcoding region (encoding the transmembrane domain and the cytoplasmictail) was obtained by inserting the synthesized fragment (SEQ ID NO: 2;FIG. 5B) previously digested with KpnI and SacI, into the H1 expressionplasmid. The resulting plasmid, named 540, is presented in FIG. 11 (alsosee FIG. 2A).

H5 A/Indonesia/5/2005 (Construct Number 663)

The signal peptide of alfalfa protein disulfide isomerase (PDISP)(nucleotides 32-103; Accession No. Z11499; SEQ ID NO: 34; FIG. 17) waslinked to the HA0 coding sequence of H5 from A/Indonesia/5/2005 asfollows. The H5 coding sequence was amplified with primersSpPDI-HA(Ind).c (SEQ ID NO:82) and HA(Ind)-SacI.r (SEQ ID NO: 7; FIG.7D) using construct number 660 (SEQ ID NO:60; FIG. 51) as template. Theresulting fragment consisted in the H5 coding sequence flanked, in 5′,by the last nucleotides encoding PDISP (including a BglII restrictionsite) and, in 3′, by a SacI restriction site. The fragment was digestedwith BglII and SacI and cloned into construct number 540 (SEQ ID NO:61;FIG. 52) previously digested with the same restriction enzymes. Thefinal cassette, named construct number 663 (SEQ ID NO:83), is presentedin FIG. 69.

H1 A/Brisbane/59/2007 (Construct 787)

The signal peptide of alfalfa protein disulfide isomerase (PDISP)(nucleotides 32-103; Accession No. Z11499; SEQ ID NO: 34; FIG. 17) waslinked to the HA0 coding sequence of H1 from A/Brisbane/59/2007 asfollows. The H1 coding sequence was amplified with primers SpPDI-H1B.c(SEQ ID NO: 84) and SacI-H1B.r (SEQ ID NO:85) using construct 774 (SEQID NO:62; FIG. 53) as template. The resulting fragment consisted in theH1 coding sequence flanked, in 5′, by the last nucleotides encodingPDISP (including a BglII restriction site) and, in 3′, by a SacIrestriction site. The fragment was digested with BglII and SacI andcloned into construct number 540 (SEQ ID NO:61; FIG. 52) previouslydigested with the same restriction enzymes. The final cassette, namedconstruct number 787 (SEQ ID NO:86), is presented in FIG. 70.

H3 A/Brisbane/10/2007 (construct number 790)

The signal peptide of alfalfa protein disulfide isomerase (PDISP)(nucleotides 32-103; Accession No. Z11499; SEQ ID NO: 34; FIG. 17) waslinked to the HA0 coding sequence of H3 from A/Brisbane/10/2007 asfollows. PDISP was linked to the H3 coding sequence by the PCR-basedligation method presented in Darveau et al. (Methods in Neuroscience 26:77-85 (1995)). In a first round of PCR, a segment of the plastocyaninepromoter fused to PDISP was amplified using primers Plasto-443c (SEQ IDNO: 4; FIG. 7A) and H3B-SpPDI.r (SEQ ID NO:87) with construct 540 (SEQID NO:61; FIG. 52) as template. In parallel, another fragment containinga portion of the coding sequence of H3 A/Brisbane/10/2007 (from codon 17to the SpeI restriction site) was amplified with primers SpPDI-H3B.c(SEQ ID NO:88) and H3(A-Bri).982r (SEQ ID NO:89) using construct 776(SEQ ID NO:69; FIG. 60) as template. Amplification products were thenmixed and used as template for a second round of amplification(assembling reaction) with primers Plasto-443c (SEQ ID NO: 4; FIG. 7A)and H13(A-Bri).982r (SEQ ID NO:89). The resulting fragment was digestedwith BamHI (in the plastocyanin promoter) and SpeI (in the H3 codingsequence) and cloned into construct number 776 (SEQ ID NO:69; FIG. 60),previously digested with the same restriction enzymes to give constructnumber 790 (SEQ ID NO:90). The construct is presented in FIG. 71.

HA B/Florida/4/2006 (Construct Number 798)

The signal peptide of alfalfa protein disulfide isomerase (PDISP)(nucleotides 32-103; Accession No. Z11499; SEQ ID NO: 34; FIG. 17) waslinked to the HA0 coding sequence of HA from HA B/Florida/4/2006 by thePCR-based ligation method presented in Darveau et al. (Methods inNeuroscience 26: 77-85 (1995)). In a first round of amplification, aportion of the plastocyanin promoter fused to the PDISP was amplifiedusing primers Plasto-443c (SEQ ID NO: 4; FIG. 7A) and HBF-SpPDI.r (SEQID NO:91) with construct number 540 (SEQ ID NO:61; FIG. 52) as template.In parallel, another fragment containing a portion of the codingsequence of HB B/Flo fused to the plastocyanin terminator was amplifiedwith primers SpPDI-HBF.c (SEQ ID NO:92) and Plaster80r (SEQ ID NO:93)using construct number 779 (SEQ ID NO:73; FIG. 64) as template. PCRproducts were then mixed and used as template for a second round ofamplification (assembling reaction) with primers Plasto-443c (SEQ ID NO:4; FIG. 7A) and Plaster80r (SEQ ID NO:93). The resulting fragment wasdigested with BamHI (in the plastocyanin promoter) and AflII (in the HAB/Florida/4/2006 coding sequence) and cloned into construct number 779(SEQ ID NO:73; FIG. 64), previously digested with the same restrictionenzymes to give construct number 798 (SEQ ID NO:94). The resultingexpression cassette is presented in FIG. 72.

Assembly of CPMV-HT-Based Expression Cassettes

CPMV-HT expression cassettes use the 35S promoter to control theexpression of an mRNA comprising a coding sequence of interest flanked,in 5′ by nucleotides 1-512 from the Cowpea mosaic virus (CPMV) RNA2 withmutated ATG at positions 115 and 161 and in 3′, by nucleotides 3330-3481from the CPMV RNA2 (corresponding to the 3′ UTR) followed by the NOSterminator. Plasmid pBD-05-1LC, (Sainsbury et al. 2008; PlantBiotechnology Journal 6: 82-92 and PCT Publication WO 2007/135480), wasused for the assembly of CPMV-H7′-based hemagglutinin expressioncassettes. The mutation of ATGs at position 115 and 161 of the CPMV RNA2was done using a PCR-based ligation method presented in Darveau et al.(Methods in Neuroscience 26: 77-85 (1995)). Two separate PCRs wereperformed using pBD-05-1LC as template. The primers for the firstamplification are pBinPlus.2613c (SEQ ID NO: 77) and Mut-ATG115.r (SEQID NO: 78). The primers for the second amplification were Mut-ATG161.c(SEQ ID NO: 79) and LC-C5-1.110r (SEQ ID NO: 80). The two obtainedfragments are then mixed and used as template for a third amplificationusing pBinPlus.2613c (SEQ ID NO: 77) and LC-05-1.110r (SEQ ID NO: 80) asprimers. Resulting fragment is digested with PacI and ApaI and clonedinto pBD-05-1LC digested with the same enzyme. The sequence of theexpression cassette generated, named 828, is presented in FIG. 68 (SEQID NO: 81).

Assembly of SpPDI-H1 A/New Caledonia/20/99 in CPMV-HT ExpressionCassette (Construct Number 580).

A sequence encoding alfalfa PDI signal peptide fused to HA0 from H1A/New Caledonia/20/99 was cloned into CPMV-HT as follows. Restrictionsites ApaI (immediately upstream of initial ATG) and StuI (immediatelydownstream stop codon) were added to the hemagglutinin coding sequenceby performing a PCR amplification with primers ApaI-SpPDI.c (SEQ ID NO:95) and StuI-H1(A-NC).r (SEQ ID NO: 96) using construct number 540 (SEQID NO:61; FIG. 52) as template. Resulting fragment was digested withApaI and StuI restriction enzymes and cloned into construct number 828(SEQ ID NO: 81) digested with the same enzymes. Resulting cassette wasnamed construct number 580 (SEQ ID NO: 97).

Assembly of H5 A/Indonesia/5/2005 in CPMV-HT Expression Cassette(Construct Number 685).

The coding sequence of H5 from A/Indonesia/5/2005 was cloned intoCPMV-HT as follows. Restriction sites ApaI (immediately upstream ATG)and StuI (immediately downstream stop codon) were added to thehemagglutinin coding sequence by performing a PCR amplification withprimers ApaI-H5 (A-Indo). 1c (SEQ ID NO: 98) and H5 (A-Indo)-StuI.1707r(SEQ ID NO: 99) using construct number 660 (SEQ ID NO:60; FIG. 51) astemplate. Resulting fragment was digested with ApaI and StuI restrictionenzymes and cloned into construct number 828 (SEQ ID NO: 81) digestedwith the same enzymes. Resulting cassette was named construct number 685(SEQ ID NO:100).

Assembly of SpPDI-H5 A/Indonesia/5/2005 in CPMV-HT Expression Cassette(Construct Number 686).

A sequence encoding alfalfa PDI signal peptide fused to HA0 from H5A/Indonesia/5/2005 was cloned into CPMV-HT as follows. Restriction sitesApaI (immediately upstream ATG) and StuI (immediately downstream stopcodon) were added to the hemagglutinin coding sequence by performing aPCR amplification with primers ApaI-SpPDI.c (SEQ ID NO: 95) and H5(A-Indo)-StuI.1707r (SEQ ID NO: 99) using construct number 663 (SEQ IDNO: 83) as template. Resulting fragment was digested with ApaI and StuIrestriction enzymes and cloned into construct number 828 (SEQ ID NO: 81)digested with the same enzymes. Resulting cassette was named constructnumber 686 (SEQ ID NO: 101).

Assembly of H1 A/Brisbane/59/2007 in CPMV-HT Expression Cassette(Construct Number 732).

The coding sequence of HA from H1 A/Brisbane/59/2007 was cloned intoCPMV-HT as follows. Restriction sites ApaI (immediately upstream ATG)and StuI (immediately downstream stop codon) were added to thehemagglutinin coding sequence by performing a PCR amplification withprimers ApaI-H₁B.c (SEQ ID NO: 102) and StuI-H1B.r (SEQ ID NO: 103)using construct number 774 (SEQ ID NO:62; FIG. 53) as template.Resulting fragment was digested with ApaI and StuI restriction enzymesand cloned into construct number 828 (SEQ ID NO: 81) digested with thesame enzymes. Resulting cassette was named construct number 732 (SEQ IDNO: 104).

Assembly of SpPDI-H1 A/Brisbane/59/2007 in CPMV-HT Expression Cassette(Construct Number 733).

A sequence encoding alfalfa PDI signal peptide fused to HA0 from H1A/Brisbane/59/2007 was cloned into CPMV-HT as follows. Restriction sitesApaI (immediately upstream ATG) and StuI (immediately downstream stopcodon) were added to the hemagglutinin coding sequence by performing aPCR amplification with primers ApaI-SpPDI.c (SEQ ID NO: 95) andStuI-H1B.r (SEQ ID NO: 103) using construct number 787 (SEQ ID NO: 86)as template. Resulting fragment was digested with ApaI and StuIrestriction enzymes and cloned into construct number 828 (SEQ ID NO: 81)digested with the same enzymes. Resulting cassette was named constructnumber 733 (SEQ ID NO: 105).

Assembly of H3 A/Brisbane/10/2007 in CPMV-HT Expression Cassette(Construct Number 735).

The coding sequence of HA from H3 A/Brisbane/10/2007 was cloned intoCPMV-HT as follows. Restriction sites ApaI (immediately upstream ATG)and StuI (immediately downstream stop codon) were added to thehemagglutinin coding sequence by performing a PCR amplification withprimers ApaI-H3B.c (SEQ ID NO:106) and StuI-H3B.r (SEQ ID NO: 107) usingconstruct number 776 (SEQ ID NO:69) as template. Resulting fragment wasdigested with ApaI and StuI restriction enzymes and cloned intoconstruct number 828 (SEQ ID NO: 81) digested with the same enzymes.Resulting cassette was named construct number 735 (SEQ ID NO: 108).

Assembly of SpPDI-H3 A/Brisbane/10/2007 in CPMV-HT Expression Cassette(Construct Number 736).

A sequence encoding alfalfa PDI signal peptide fused to HA0 from H3A/Brisbane/10/2007 was cloned into CPMV-HT as follows. Restriction sitesApaI (immediately upstream ATG) and StuI (immediately downstream stopcodon) were added to the hemagglutinin coding sequence by performing aPCR amplification with primers ApaI-SpPDI.c (SEQ ID NO:95) andStuI-H3B.r (SEQ ID NO: 107) using construct number 790 (SEQ ID NO:90) astemplate. Resulting fragment was digested with ApaI and StuI restrictionenzymes and cloned into construct number 828 (SEQ ID NO: 81) digestedwith the same enzymes. Resulting cassette was named construct number 736(SEQ ID NO:109).

Assembly of HA B/Florida/4/2006 in CPMV-HT Expression Cassette(Construct Number 738).

The coding sequence of HA from B/Florida/4/2006 was cloned into CPMV-HTas follows. Restriction sites ApaI (immediately upstream ATG) and StuI(immediately downstream stop codon) were added to the hemagglutinincoding sequence by performing a PCR amplification with primersApaI-HBF.c (SEQ ID NO: 110) and StuI-HBF.r (SEQ ID NO: 111) usingconstruct number 779 (SEQ ID NO:73; FIG. 64) as template. Resultingfragment was digested with ApaI and StuI restriction enzymes and clonedinto construct number 828 (SEQ ID NO: 81) digested with the sameenzymes. Resulting cassette was named construct number 738 (SEQ ID NO:112).

Assembly of SpPDI-HA B/Florida/4/2006 in CPMV-HT Expression Cassette(Construct Number 739).

A sequence encoding alfalfa PDI signal peptide fused to HA0 fromB/Florida/4/2006 was cloned into CPMV-HT as follows. Restriction sitesApaI (immediately upstream ATG) and StuI (immediately downstream stopcodon) were added to the hemagglutinin coding sequence by performing aPCR amplification with primers ApaI-SpPDI.c (SEQ ID NO: 95) andStuI-HBF.r (SEQ ID NO: 111) using construct number 798 (SEQ ID NO: 94)as template. Resulting fragment was digested with ApaI and StuIrestriction enzymes and cloned into construct number 828 (SEQ ID NO: 81)digested with the same enzymes. Resulting cassette was named constructnumber 739 (SEQ ID NO: 113).

Assembly of Chaperone Expression Cassettes

Two heat shock protein (Hsp) expression cassettes were assembled. In afirst cassette, expression of the Arabidopsis thaliana (ecotypeColumbia) cytosolic HSP70 (Athsp70-1 in Lin et al. (2001) Cell Stressand Chaperones 6: 201-208) is controlled by a chimeric promotercombining elments of the alfalfa Nitrite reductase (Nir) and alfalfaPlastocyanin promoters (Nir/Plasto). A second cassette comprising thecoding region of the alfalfa cytosolic HSP40 (MsJ1; Frugis et al. (1999)Plant Molecular Biology 40: 397-408) under the control of the chimericNir/Plasto promoter was also assembled.

An acceptor plasmid containing the alfalfa Nitrite reductase promoter(Nir), the GUS reporter gene and NOS terminator in plant binary vectorwas first assembled. Plasmid pNir3K51 (previously described in U.S. Pat.No. 6,420,548) was digested with HindIII and EcoRI. The resultingfragment was cloned into pCAMBIA2300 (Cambia, Canberra, Australia)digested by the same restriction enzyme to give pCAMBIA-Nir3K51.

Coding sequences for Hsp70 and Hsp40 were cloned separately in theacceptor plasmid pCAMBIANir3K51 by the PCR-based ligation methodpresented in Darveau et al. (Methods in Neuroscience 26:77-85 (1995)).

For Hsp40, Msj1 coding sequence (SEQ ID NO: 114) was amplified by RT-PCRfrom alfalfa (ecotype Rangelander) leaf total RNA using primersHsp40Luz.1c (SEQ ID NO: 115) and Hsp40Luz-SacI.1272r (SEQ ID NO: 116). Asecond amplification was performed with primers Plasto-443c (SEQ ID NO:4; FIG. 7A) and Hsp40Luz-Plasto.r (SEQ ID NO: 117) with construct 660(SEQ ID NO: 60; FIG. 51) as template. PCR products were then mixed andused as template for a third amplification (assembling reaction) withprimers Plasto-443c (SEQ ID NO: 4; FIG. 7A) and Hsp40Luz-SacI.1272r (SEQID NO: 116). The resulting fragment was digested with HpaI (in theplastocyanin promoter) and cloned into pCAMBIANir3K51, previouslydigested with HpaI (in the Nir promoter) and SacI, and filed with T4 DNApolymerase to generate blunt ends. Clones obtained were screened forcorrect orientation and sequenced for sequence integrity. The resultingplasmid, named R850, is presented in FIG. 83 (SEQ ID NO: 121). Thecoding region of the Athsp70-1 was amplified by RT-PCR from Arabidopsisleaf RNA using primers Hsp70Ara.1c (SEQ ID NO: 118) andHsp70Ara-SacI.1956r (SEQ ID NO: 119). A second amplification wasperformed with primers Plato-443c (SEQ ID NO: 4; FIG. 7A) andHsp70Ara-Plasto.r (SEQ ID NO: 120) with construct 660 (SEQ ID NO: 60;FIG. 51) as template. PCR products were then mixed and used as templatefor a third amplification (assembling reaction) with primers Plasto-443c(SEQ ID NO: 4; FIG. 7A) and Hsp70ARA-SacI.1956r (SEQ ID NO: 119). Theresulting fragment was digested with HpaI (in the plastocyanin promoter)and cloned into pCAMBIANir3K51 digested with HpaI (in the Nir promoter)and SacI and filed with T4 DNA polymerase to generate blunt ends. Clonesobtained were screened for correct orientation and sequenced forsequence integrity. The resulting plasmid, named R860, is presented inFIG. 84 (SEQ ID NO: 122).

A dual Hsp expression plasmid was assembled as follows. R860 wasdigested with BsrBI (downstream the NOS terminator), treated with T4 DNApolymerase to generate a blunt end, and digested with SbfI (upstream thechimeric NIR/Plasto promoter). The resulting fragment (ChimericNir/Plasto promoter-HSP70 coding sequence-Nos terminator) was clonedinto R850 previously digested with SbfI and SmaI (both located in themultiple cloning site upstream chimeric Nir/Plasto promoter). Theresulting plasmid, named R870, is presented in FIG. 85 (SEQ ID NO: 123).

Assembly of Other Expression Cassettes

Soluble H1 Expression Cassette

The cassette encoding the soluble form of H1 was prepared by replacingthe region coding for the transmembrane domain and the cytoplasmic tailin 540 by a fragment encoding the leucine zipper GCN4 pII variant(Harbury et al, 1993, Science 1993; 262: 1401-1407). This fragment wassynthesized with flanking KpnI and SacI sites to facilitate cloning. Theplasmid resulting from this replacement was named 544 and the expressioncassette is illustrated in FIG. 11.

M1 A/Puerto Rico/8/34 Expression Cassette

A fusion between the tobacco etch virus (TEV) 5′UTR and the open readingframe of the influenza A/PR/8/34 M1 gene (Acc. #NC_002016) wassynthesized with a flanking SacI site added downstream of the stopcodon. The fragment was digested with SwaI (in the TEV 5′UTR) and SacI,and cloned into a 2×35S/TEV based expression cassette in a pCAMBIAbinary plasmid. The resulting plasmid bore the M1 coding region underthe control of a 2×35S/TEV promoter and 5′UTR and the NOS terminator(construct 750; FIG. 11).

HcPro Expression Cassette

An HcPro construct (35HcPro) was prepared as described in Hamilton etal. (2002). All clones were sequenced to confirm the integrity of theconstructs. The plasmids were used to transform Agrobacteium tumefaciens(AGL1; ATCC, Manassas, Va. 20108, USA) by electroporation (Mattanovichet al., 1989). The integrity of all A. tumefaciens strains wereconfirmed by restriction mapping.

P19 Expression Cassette

The coding sequence of p19 protein of tomato bushy stunt virus (TBSV)was linked to the alfalfa plastocyanin expression cassette by thePCR-based ligation method presented in Darveau et al. (Methods inNeuroscience 26: 77-85 (1995)). In a first round of PCR, a segment ofthe plastocyanin promoter was amplified using primers Plasto-443c (SEQID NO: 4; FIG. 7A) and supP19-plasto.r (SEQ ID NO:124) with construct660 (SEQ ID NO:60; FIG. 51) as template. In parallel, another fragmentcontaining the coding sequence of p19 was amplified with primerssupP19-1c (SEQ ID NO:125) and SupP19-SacI.r (SEQ ID NO: 126) usingconstruct 35S:p19 as described in Voinnet et al. (The Plant Journal 33:949-956 (2003)) as template. Amplification products were then mixed andused as template for a second round of amplification (assemblingreaction) with primers Plasto-443c (SEQ ID NO: 4; FIG. 7A) andSupP19-SacI.r (SEQ ID NO: 126). The resulting fragment was digested withBamHI (in the plastocyanin promoter) and SacI (at the end of the p19coding sequence) and cloned into construct number 660 (SEQ ID NO:60;FIG. 51), previously digested with the same restriction enzymes to giveconstruct number R472. Plasmid R472 is presented in FIG. 86.

3. Preparation of Plant Biomass, Inoculum, Agroinfiltration, andHarvesting

Nicotiana benthamiana or Nicotiana tabacum plants were grown from seedsin flats filled with a commercial peat moss substrate. The plants wereallowed to grow in the greenhouse under a 16/8 photoperiod and atemperature regime of 25° C. day/20° C. night. Three weeks afterseeding, individual plantlets were picked out, transplanted in pots andleft to grow in the greenhouse for three additional weeks under the sameenvironmental conditions. Prior to transformation, apical and axillarybuds were removed at various times as indicated below, either bypinching the buds from the plant, or by chemically treating the plant

Agrobacteria transfected with each construct were grown in a YEB mediumsupplemented with 10 mM 2-[N-morpholino]ethanesulfonic acid (MES), 20 μMacetosyringone, 50 μg/ml kanamycin and 25 μg/ml of carbenicillin pH5.6until they reached an OD₆₀₀ between 0.6 and 1.6. Agrobacteriumsuspensions were centrifuged before use and resuspended in infiltrationmedium (10 mM MgCl₂ and 10 mM MES pH 5.6). Syringe-infiltration wasperformed as described by Liu and Lomonossoff (2002, Journal ofVirological Methods, 105:343-348). For vacuum-infiltration, A.tumefaciens suspensions were centrifuged, resuspended in theinfiltration medium and stored overnight at 4° C. On the day ofinfiltration, culture batches were diluted in 2.5 culture volumes andallowed to warm before use. Whole plants of N. benthamiana or N. tabacumwere placed upside down in the bacterial suspension in an air-tightstainless steel tank under a vacuum of 20-40 Torr for 2-min. Followingsyringe or vacuum infiltration, plants were returned to the greenhousefor a 4-5 day incubation period until harvest. Unless otherwisespecified, all infiltrations were performed as co-infiltration withAGL1/35S-HcPro in a 1:1 ratio, except for CPMV-HT cassette-bearingstrains which were co-infiltrated with strain AGL1/R472 in a 1:1 ratio.

4. Leaf Sampling and Total Protein Extraction

Following incubation, the aerial part of plants was harvested, frozen at−80° C., crushed into pieces. Total soluble proteins were extracted byhomogenizing (Polytron) each sample of frozen-crushed plant material in3 volumes of cold 50 mM Tris pH 7.4, 0.15 M NaCl, and 1 mMphenylmethanesulfonyl fluoride. After homogenization, the slurries werecentrifuged at 20,000 g for 20 min at 4° C. and these clarified crudeextracts (supernatant) kept for analyses. The total protein content ofclarified crude extracts was determined by the Bradford assay (Bio-Rad,Hercules, Calif.) using bovine serum albumin as the reference standard.

5. Size Exclusion Chromatography of Protein Extract

Size exclusion chromatography (SEC) columns of 32 ml Sephacryl™ S-500high resolution beads (S-500 HR:GE Healthcare, Uppsala, Sweden, Cat. No.17-0613-10) were packed and equilibrated with equilibration/elutionbuffer (50 mM Tris pH8, 150 mM NaCl). One and a half milliliter of crudeprotein extract was loaded onto the column followed by an elution stepwith 45 mL of equilibration/elution buffer. The elution was collected infractions of 1.5 mL relative protein content of eluted fractions wasmonitored by mixing 10 μL of the fraction with 200 μL of diluted Bio-Radprotein dye reagent (Bio-Rad, Hercules, Calif. The column was washedwith 2 column volumes of 0.2N NaOH followed by 10 column volumes of 50mM Tris p118, 150 mM NaCl, 20% ethanol. Each separation was followed bya calibration of the column with Blue Dextran 2000 (GE HealthcareBio-Science Corp., Piscataway, N.J., USA). Elution profiles of BlueDextran 2000 and host soluble proteins were compared between eachseparation to ensure uniformity of the elution profiles between thecolumns used.

6. Protein Analysis and Immunoblotting

Protein concentrations were determined by the BCA protein assay (PierceBiochemicals, Rockport Ill.). Proteins were separated by SDS-PAGE underreducing conditions and stained with Coomassie Blue. Stained gels werescanned and densitometry analysis performed using ImageJ Software (NIH).

Proteins from elution fraction from SEC were precipitated with acetone(Bollag et al., 1996), resuspended in 1/5 volume inequilibration/elution buffer and separated by SDS-PAGE under reducingconditions and electrotransferred onto polyvinylene difluoride (PVDF)membranes (Roche Diagnostics Corporation, Indianapolis, Ind.) forimmunodetection. Prior to immunoblotting, the membranes were blockedwith 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T) for16-18 h at 4° C.

Immunoblotting was performed by incubation with a suitable antibody(Table 6), in 2 μg/ml in 2% skim milk in TBS-Tween 20 0.1%. Secondaryantibodies used for chemiluminescence detection were as indicated inTable 4, diluted as indicated in 2% skim milk in TBS-Tween 20 0.1%.Immunoreactive complexes were detected by chemiluminescence usingluminol as the substrate (Roche Diagnostics Corporation). Horseradishperoxidase-enzyme conjugation of human IgG antibody was carried out byusing the EZ-Link Plus® Activated Peroxidase conjugation kit (Pierce,Rockford, Ill.). Whole, inactivated virus (WIV), used as controls ofdetection for H1, H3 and B subtypes, were purchased from NationalInstitute for Biological Standards and Control (NIBSC).

TABLE 6 Electrophoresis conditions, antibodies, and dilutions forimmunoblotting of expressed proteins. HA Electrophoresis PrimarySecondary subtype Influenza strain condition antibody Dilution antibodyDilution H1 A/Brisbane/59/2007 Reducing FII 4 μg/ml Goat anti-mouse 1:10000 (H1N1) 10-I50 (JIR 115-035-146) H1 A/Solomon Islands/ Reducing NIBSC1:2000 Rabbit anti-sheep 1:10 000 3/2006 (H1N1) 07/104 (JIR 313-035-045)H1 A/New Caledonia/ Reducing FII 4 μg/ml Goat anti-mouse 1:10 000 20/99(H1N1) 10-I50 (JIR 115-035-146) H2 A/Singapore/1/57 Non-reducing NIBSC1:1000 Rabbit anti-sheep 1:10 000 (H2N2) 00/440 (JIR 313-035-045) H3A/Brisbane/10/ Non-Reducing TGA 1:4000 Rabbit anti-sheep 1:10 000 2007(H3N2) AS393 (JIR 313-035-045) H3 A/Brisbane/10/ Non-Reducing NIBSC1:1000 Rabbit anti-sheep 1:10 000 2007 (H3N2) 08/136 (JIR 313-035-045)H3 A/Wisconsin/67/ Non-Reducing NIBSC 1:1000 Rabbit anti-sheep 1:10 0002005 (H3N2) 05/236 (JIR 313-035-045) H5 A/Indonesia/5/ Reducing ITC IT-1:4000 Goat anti-rabbit 1:10 000 2005 (H5N1) 003-005V (JIR 111-035-144)H5 A/Anhui/1/2005 Reducing NIBSC 1:750  Rabbit anti-sheep 1:10 000(H5N1) 07/338 (JIR 313-035-045) H5 A/Vietnam/1194/ Non-reducing ITC IT-1:2000 Goat anti-rabbit 1:10 000 2004 (H5N1) 003-005 (JIR 111-035-144)H6 A/Teal/Hong Kong/ Non-reducing BEI NR 1:500  Rabbit anti-sheep 1:10000 W312/97 (H6N1) 663 (JIR 313-035-045) H7 A/Equine/Prague/Non-reducing NIBSC 1:1000 Rabbit anti-sheep 1:10 000 56 (H7N7) 02/294(JIR 313-035-045) H9 A/Hong Kong/ Reducing NIBSC 1:1000 Rabbitanti-sheep 1:10 000 1073/99 (H9N2) 07/146 (JIR 313-035-045) BB/Malaysia/2506/ Non-Reducing NIBSC 1:2000 Rabbit anti-sheep 1:10 0002004 07/184 (JIR 313-035-045) B B/Florida/4/2006 Non-Reducing NIBSC1:2000 Rabbit anti-sheep 1:10 000 07/356 (JIR 313-035-045) FII:Fitzgerald Industries International, Concord, MA, USA; NIBSC: NationalInstitute for Biological Standards and Control; JIR: JacksonImmunoResearch, West Grove, PA, USA; BEI NR: Biodefense and emerginginfections research resources repository; ITC: Immune TechnologyCorporation, Woodside, NY, USA; TGA: Therapeutic Goods Administration,Australia.

Hemagglutination assay for H5 was based on a method described by Nayakand Reichl (2004). Briefly, serial double dilutions of the test samples(100 μl) were made in V-bottomed 96-well microtiter plates containing100 μL PBS, leaving 100 μL of diluted sample per well. One hundredmicroliters of a 0.25% turkey red blood cells suspension (Bio Link Inc.,Syracuse, N.Y.) were added to each well, and plates were incubated for 2h at room temperature. The reciprocal of the highest dilution showingcomplete hemagglutination was recorded as HA activity. In parallel, arecombinant HA standard (A/Vietnam/1203/2004 H5N1) (Protein ScienceCorporation, Meriden, Conn.) was diluted in PBS and run as a control oneach plate.

7. Sucrose Gradient Ultracentrifugation

One milliliter of fractions 9, 10 and 11 eluted from the gel filtrationchromatography on H5-containing biomass were pooled, loaded onto a20-60% (w/v) discontinuous sucrose density gradient, and centrifuged17.5 h at 125 000 g (4° C.). The gradient was fractionated in 19 3-mLfractions starting from the top, and dialyzed to remove sucrose prior toimmunological analysis and hemagglutination assays.

8. Electron Microscopy

One hundred microliters of the samples to be examined were placed in anAirfuge ultracentrifugation tube (Beckman Instruments, Palo Alto,Calif., USA). A grid was placed at the bottom of the tube which was thencentrifuged 5 min at 120 000 g. The grid was removed, gently dried, andplaced on a drop of 3% phosphotungstic acid at pH 6 for staining. Gridswere examined on a Hitachi 7100 transmission electron microscope (TEM)(for images in FIGS. 14B, 15B and 15C).

For images in FIG. 19, Leaf blocks of approximately 1 mm³ were fixed inPBS containing 2.5% glutaraldehyde and washed in PBS containing 3%sucrose before a post-fixation step in 1.33% osmium tetroxide. Fixedsamples were imbedded in Spurr resin and ultrathin layers were laid on agrid. Samples were positively stained with 5% uranyl acetate and 0.2%lead citrate before observation. Grids were examined on a Hitachi 7100transmission electron microscope (TEM).

9. Plasma Membrane Lipid Analysis

Plasma membranes (PM) were obtained from tobacco leaves and cultured BY2cells after cell fractionation according to Mongrand et any partitioningin an aqueous polymer two-phase system with polyethylene glycol3350/dextran T-500 (6.6% each). All steps were performed at 4° C.

Lipids were extracted and purified from the different fractionsaccording to Bligh and Dyer. Polar and neutral lipids were separated bymono-dimensional HP-TLC using the solvent systems described in Lefebvreet al. Lipids of PM fractions were detected after staining with copperacetate as described by Macala et al. Lipids were identified bycomparison of their migration time with those of standards (allstandards were obtained from Sigma-Aldrich, St-Louis, Mo., USA, exceptfor SG which was obtained from Matreya, Pleasant Gap, Pa., USA).

10. H5 VLP (A/Indonesia/5/2005) Purification

Frozen 660-infiltrated leaves of N. benthamiana were homogenized in 1.5volumes of 50 mM Tris pH 8, NaCl 150 mM and 0.04% sodium meta-bisulfiteusing a commercial blender. The resulting extract was supplemented with1 mM PMSF and adjusted to pH 6 with 1 M acetic acid before being heatedat 42° C. for 5 min. Diatomaceous earth (DE) was added to theheat-treated extract to adsorb the contaminants precipitated by the pHshift and heat treatment, and the slurry was filtered through a Whatmanpaper filter. The resulting clarified extract was centrifuged at10,000×g for 10 minutes at RT to remove residual DE, passed through0.8/0.2 μm Acropack 20 filters and loaded onto a fetuin-agarose affinitycolumn (Sigma-Aldrich, St-Louis, Mo., USA). Following a wash step in 400mM NaCl, 25 mM Tris pH 6, bound proteins were eluted with 1.5 M NaCl, 50mM MES pH 6. Eluted VLP were supplemented with Tween-80 to a finalconcentration of 0.0005% (v/v). VLP were concentrated on a 100 kDa MWCOAmicon membrane, centrifuged at 10,000×g for 30 minutes at 4° C. andresuspended in PBS pH 7.4 with 0.01% Tween-80 and 0.01% thimerosal.Suspended VLPs were filter-sterilized before use.

11. Animal Studies

Mice

Studies on the immune response to influenza VLP administration wereperformed with 6-8 week old female BALB/c mice (Charles RiverLaboratories). Seventy mice were randomly divided into fourteen groupsof five animals. Eight groups were used for intramuscular immunizationand six groups were used to test intranasal route of administration. Allgroups were immunized in a two-dose regiment, the boost immunizationbeing done 3 weeks following the first immunization.

For intramuscular administration in hind legs, unanaesthetized mice wereimmunized with either the plant-made H5 VLP (A/Indonesia/5/2005 (H5N1)vaccine (0.1, 1, 5 or 12 μg), or a control hemagglutinin (H5) antigen.The control H5 comprised recombinant soluble hemagglutinin producedbased on strain A/Indonesia/5/05 H5N1 and purified from 293 cell culture(Immune Technology Corp., New York, USA) (used at 5 μg per injectionunless otherwise indicated). Buffer control was PBS. This antigenconsists of amino acids 18-530 of the HA protein, and has a His-tag anda modified cleavage site. Electron microscopy confirmed that thiscommercial product is not in the form of VLPs.

To measure the effect of adjuvant, two groups of animals were immunizedwith 5 μg plant-made VLP H5 vaccine plus one volume Alhydrogel 2% (alum,Accurate Chemical & Scientific Corporation, Westbury, N.Y., US) or with5 μg recombinant hemagglutinin purified from 293 cell culture plus 1volume alum. Seventy mice were randomly divided into fourteen groups offive animals. Eight groups were used for intramuscular immunization andsix groups were used to test intranasal route of administration. Allgroups were immunized according to a prime-boost regimen, the boostimmunization performed 3 weeks following the first immunization.

For intramuscular administration in hind legs, unanaesthetized mice wereimmunized with the plant-made VLP (0.1, 1, 5 or 12 μg), or the controlhemagglutinin (HA) antigen (5 μg) or PBS. All antigen preparations weremixed with Alhydrogel 1% (alum, Accurate Chemical & ScientificCorporation, Westbury, N.Y., US) in a 1:1 volume ratio prior toimmunizations. To measure the effect of adjuvant, two groups of animalswere immunized with either 5 μg plant-made VLP H5 vaccine or with 5 μgof control HA antigen without any adjuvant.

For intranasal administration, mice were briefly anaesthetized byinhalation of isoflurane using an automated induction chamber. They werethen immunized by addition of 4 μl drop/nostril with the plant-made VLPvaccine (0.1 or 1 μg), or with control HA antigen (1 μg) or with PBS.All antigen preparations were mixed with chitosan glutamate 1%(Protosan, Novamatrix/FMC BioPolymer, Norway) prior to immunizations.The mice then breathed in the solutions. To verify the effect ofadjuvant with the intranasal route of administration, two groups ofanimals were immunized with 1 μg plant-made VLP H5 vaccine or with 1 μgcontrol HA antigen.

Ferrets

Ten groups of 5 ferrets (male, 18-24 weeks old, mass of approx 1 kg)were used. Treatment for each group is as described in Table 7. Theadjuvant used was Alhydrogel (alum) (Superfos Biosector, Denmark) 2%(final=1%). Vaccine composition was membrane-associated A/Indonesia/5/05(H5N1) VLPs produced as described. The vaccine control (positivecontrol) was a fully glycosylated membrane-bound recombinant H5 fromIndonesia strain produced using adenovirus in 293 cell culture by ImmuneTechnology Corporation (ITC).

TABLE 7 Treatment groups Product injected to Route of Group n animalsadministration Adjuvant 1 5 PBS (negative control) i.m.* — 2 5Vaccine-plant, 1 μg i.m. — 3 5 Vaccine-plant, 1 μg i.m. Alum 4 5Vaccine-plant, 5 μg i.m. — 5 5 Vaccine-plant, 5 μg i.m. Alum 6 5Vaccine-plant, 7.5 μg i.m. — 7 5 Vaccine-plant, 15 μg i.m. — 8 5Vaccine-plant, 15 μg i.m. Alum 9 5 Vaccine-plant, 30 μg i.m. — 10 5Vaccine-control, 5 μg i.m. — *i.m.: intramuscular

Ferrets were assessed for overall health and appearance (body weight,rectal temperature, posture, fur, movement patterns, breathing,excrement) regularly during the study. Animals were immunized byintramuscular injection (0.5-1.0 total volume) in quadriceps at day 0,14 and 28; for protocols incorporating adjuvant, the vaccine compositionwas combined with Alhydrogel immediately prior to immunization in a 1:1volume ratio). Serum samples were obtained on day 0 before immunizing,and on day 21 and 35. Animals were sacrificed (exsanguination/cardiacpuncture) on days 40-45, and, spleens were collected and necropsyperformed.

Anti-influenza antibody titres may be quantified in ELISA assays usinghomologous or heterologous inactivated H5N1 viruses.

Hemagglutination inhibitory antibody titers of serum samples(pre-immune, day 21 and day 35) were evaluated by microtiter HAI asdescribed (Aymard et al 1973). Briefly, sera were pretreated withreceptor-destroying enzyme, heat-inactivated and mixed with a suspensionof erythrocytes (washed red blood cells-RBC). Horse washed RBC (10%)from Lampire are recommended and considering that the assay may varydepending of the source of the RBC (horse-dependant), washed RBCs from10 horses have been tested to select the most sensitive batch.Alternately, turkey RBC may be used. Antibody titer was expressed as thereciprocal of the highest dilution which completely inhibitshemagglutination.

Cross-reactive HAI titers: HAI titers of ferrets immunized with avaccine for the A/Indonesia/5/05 (Glade 2.1) were measured usinginactivated H5N1 influenza strains from another subclade or Glade suchas the Glade 1 Vietnam strains A/Vietnam/1203/2004 andA/Vietnam/1194/2004 or the A/Anhui/01/2005 (subclade 2.3) or theA/turkey/Turkey/1/05 (subclade 2.2). All analyses were performed onindividual samples.

Data analysis: Statistical analysis (ANOVA) were performed on all datato establish if differences between groups are statisticallysignificant.

Experimental Design for Lethal Challenge (Mice)

One hundred twenty eight mice were randomly divided into sixteen groupsof eight animals, one group being unimmunized and not challenged(negative control). All groups were immunized via intramuscularadministration in a two-dose regimen, the second immunization being done2 weeks following the first immunization.

For intramuscular administration in hind legs, unanaesthetized mice wereimmunized with the plant-made H5 VLP (1, 5 or 15 μg), or 15 μg ofcontrol HA antigen or PBS. All antigen preparations were mixed with onevolume of Alhydrogel 1% prior to immunizations (alum, Accurate Chemical& Scientific Corporation, Westbury, N.Y., US).

During the immunization period, mice were weighted once a week andobservation and monitored for local reactions at the injection site.

Twenty two days following the second immunization, anesthetized micewere challenged intranasally (i.n.) into a BL4 containment laboratory(P4-Jean Mérieux-INSERM, Lyon, France) with 4.09×10⁶ 50% cell cultureinfective dose (CClD50) of influenza A/Turkey/582/06 virus (kindlyprovided by Dr. Bruno Lina, Lyon University, Lyon, France). Followingchallenge, mice were observed for ill clinical symptoms and weigheddaily, over a fourteen day period. Mice with severe infection symptomsand weight loss of ≧25% were euthanized after anaesthesia.

Blood Collection, Lung and Nasal Washes and Spleen Collection

Lateral saphenous vein blood collection was performed fourteen daysafter the first immunization and fourteen days after second immunizationon unanaesthetized animal. Serum was collected by centrifugation at 8000g for 10 min.

Four weeks after second immunisation, mice were anaesthetized with CO₂gas and immediately upon termination, cardiac puncture was used tocollect blood.

After final bleeding, a catheter was inserted into the trachea towardsthe lungs and one ml of cold PBS-protease inhibitor cocktail solutionwas put into a ice syringe attached to the catheter and injected intothe lungs and then removed for analysis. This wash procedure wasperformed two times. The lung washes were centrifuged to remove cellulardebris. For nasal washes, a catheter was inserted towards the nasal areaand 0.5 ml of the PBS-protease inhibitor cocktail solution was pushedthrough the catheter into the nasal passages and then collected. Thenasal washes were centrifuged to remove cellular debris. Spleencollection was performed on mice immunized intramuscularly with 5 μg ofadjuvanted plant-made vaccine or 5 μg adjuvanted recombinant 115 antigenas well as on mice immunized intranasaly with 1 μg of adjuvantedplant-made vaccine or 1 μg adjuvanted recombinant H5 antigen. Collectedspleens were placed in RPMI supplemented with gentamycin and mashed in a50 ml conical tube with plunger from a 10 ml syringe. Mashed spleenswere rinsed 2 times and centrifuged at 2000 rpm for 5 min andresuspended in ACK lysing buffer for 5 min at room temperature. Thesplenocytes were washed in PBS-gentamycin, resuspended in 5% RPMI andcounted. Splenocytes were used for proliferation assay.

Antibody Titers

Anti-influenza antibody titers of sera were measured at 14 days afterthe first immunization as well as 14 and 28 days after the secondimmunisation. The titer were determined by enzyme-linked immunosorbentassay (ELISA) using the inactivated virus A/Indonesia/5/05 as thecoating antigen. The end-point titers were expressed as the reciprocalvalue of the highest dilution that reached an OD value of at least 0.1higher than that of negative control samples.

For antibody class determination (IgG1, IgG2a, IgG2b, IgG3, IgM), thetiters were evaluated by ELISA as previously described.

Hemagglutination Inhibition (HI) Titers

Hemagglutination inhibition (HI) titers of sera were measured at 14 and28 days after the second immunisation as previously described (WHO 2002;Kendal 1982). Inactivated virus preparations from strainsA/Indonesia/5/05 or A/Vietnam/1203/2004 were used to test mouse serumsamples for HI activity. Sera were pre-treated with receptor-destroyingenzyme II (RDE II) (Denka Seiken Co., Tokyo, Japan) prepared from Vibriocholerae (Kendal 1982). HI assays were performed with 0.5% turkey redblood cells. HI antibody titres were defined as the reciprocal of thehighest dilution causing complete inhibition of agglutination.

EXAMPLES Example 1 Transient Expression of Influenza VirusA/Indonesia/5/05 (H5N1) Hemagglutinin by Agroinfiltration in N.benthamiana Plants

The ability of the transient expression system to produce influenzahemagglutinin was determined through the expression of the H5 subtypefrom strain A/Indonesia/5/05 (H5N1). As presented in FIG. 11, thehemagglutinin gene coding sequence (GenBank Accession No. EF541394),with its native signal peptide and transmembrane domain, was firstassembled in the plastocyanin expression cassette—promoter, 5′UTR, 3′UTRand transcription termination sequences from the alfalfa plastocyaningene—and the assembled cassette (660) was inserted into to a pCAMBIAbinary plasmid. This plasmid was then transfected into Agrobacterium(AGL1), creating the recombinant strain AGL1/660, which was used fortransient expression.

N. benthamiana plants were infiltrated with AGL1/660, and the leaveswere harvested after a six-day incubation period. To determine whetherH5 accumulated in the agroinfiltrated leaves, protein were firstextracted from infiltrated leaf tissue and analyzed by Western blottingusing anti-H5 (Vietnam) polyclonal antibodies. A unique band ofapproximately 72 kDa was detected in extracts (FIG. 12), correspondingin size to the uncleaved HA0 form of influenza hemagglutinin. Thecommercial H5 used as positive control (A/Vietnam/1203/2004; ProteinScience Corp., Meriden, Conn., USA) was detected as two bands ofapproximately 48 and 28 kDa, corresponding to the molecular weight ofHA1 and HA2 fragments, respectively. This demonstrated that expressionof H5 in infiltrated leaves results in the accumulation of the uncleavedtranslation product.

The formation of active HA trimers was demonstrated by the capacity ofcrude protein extracts from AGL1/660-transformed leaves to agglutinateturkey red blood cells (data not shown).

Example 2 Characterization of Hemagglutinin-Containing Structures inPlant Extracts Using Size Exclusion Chromatography

The assembly of plant-produced influenza hemagglutinin into highmolecular weight structures was assessed by gel filtration. Crudeprotein extracts from AGL1/660-infiltrated plants (1.5 mL) werefractionated by size exclusion chromatography (SEC) on Sephacryl™ S-500HR columns (GE Healthcare Bio-Science Corp., Piscataway, N.J., USA).Elution fractions were assayed for their total protein content and forHA abundance using immunodetection with anti-HA antibodies (FIG. 13A).As shown in FIG. 13A, Blue Dextran (2 MDa) elution peaked early infraction 10 while the bulk of host proteins was retained in the columnand eluted between fractions 14 and 22. When proteins from 200 μl, ofeach SEC elution fraction were concentrated (5-fold) byacetone-precipitation and analyzed by Western blotting (FIG. 15A, H5),hemagglutinin (H5) was primarily found in fractions 9 to 14 (FIG. 13B).Without wishing to be bound by theory, this suggests that the HA proteinhad either assembled into a large superstructure or that it has attachedto a high molecular weight structure.

A second expression cassette was assembled with the H1 nucleic acidsequence from A/New Caledonia/20/99 (H1N1) (SEQ ID NO: 33; FIG. 16;GenBank Accession No. AY289929) to produce construct 540 (FIG. 11). Achimeric gene construct was designed so as to produce a soluble trimericform of H1 in which the signal peptide originated from a plant proteindisulfide isomerase gene, and the transmembrane domain of H1 wasreplaced by the pII variant of the GCN4 leucine zipper, a peptide shownto self-assemble into trimers (Harbury et al., 1993) (cassette 544, FIG.11). Although lacking the transmembrane domain, this soluble trimericform was capable of hemagglutination (data not shown).

Protein extracts from plants infiltrated with AGL1/540 or AGL1/544 werefractionated by SEC and the presence of H1 eluted fractions was examinedby Western blotting with anti-influenza A antibodies (Fitzgerald,Concord, Mass., USA). In AGL1/540-infiltrated leaves, H1 accumulatedmainly as a very high molecular weight structure, with the peak wasskewed toward smaller size structures (H1; FIG. 13C). InAGL1/544-infiltrated leaves, the soluble form of H1 accumulated asisolated trimers as demonstrated by the elution pattern from gelfiltration which parallels the host protein elution profile (soluble H1;FIG. 13D). In comparison, H1 rosettes (Protein Science Corp., Meriden,Conn., USA), consisting in micelles of 5-6 trimers of hemagglutinineluted at fractions 12 to 16 (FIG. 13E), earlier than the soluble formof H1 (FIG. 13D) and later than the native H1 (FIG. 13C).

To evaluate the impact of M1 co-expression on hemagglutinin assemblyinto structure, a M1 expression cassette was assembled using the nucleicacid corresponding to the coding sequence of the A/PR/8/34 (H1N1) M1(SEQ ID NO: 35; FIG. 18; GenBank Accession No. NC_002016). The constructwas named 750 and is presented in FIG. 11. For the co-expression of M1and H1, suspensions of AGL1/540 and AGL1/750 were mixed in equal volumebefore infiltration. Co-infiltration of multiple Agrobacteriumsuspensions permits co-expression of multiple transgenes. The Westernblot analysis of SEC elution fractions shows that the co-expression ofM1 did not modify the elution profile of the H1 structures, but resultedin a decrease in H1 accumulation in the agroinfiltrated leaves (see FIG.13F).

Example 3 Isolation of H5 Structures by Centrifugation in SucroseGradient and Observation Under Electron Microscopy

The observation of hemagglutinin structure under electron microscopy(EM) required a higher concentration and purity level than that obtainedfrom SEC on crude leaf protein extracts. To allow EM observation of H5structures, a crude leaf protein extract was first concentrated by PEGprecipitation (20% PEG) followed by resuspension in 1/10 volumes ofextraction buffer. The concentrated protein extract was fractionated byS-500 HR gel filtration and elution fractions 9, 10, and 11(corresponding to the void volume of the column) were pooled and furtherisolated from host proteins by ultracentrifugation on a 20-60% sucrosedensity gradient. The sucrose gradient was fractionated starting fromthe top and the fractions were dialysed and concentrated on a 100 NMWLcentrifugal filter unit prior to analysis. As shown on the Western blotsand hemagglutination results (FIG. 14A), H5 accumulated mainly infractions 16 to 19 which contained ≈60% sucrose, whereas most of thehost proteins peaked at fraction 13. Fractions 17, 18, and 19 werepooled, negatively stained, and observed under EM. Examination of thesample clearly demonstrated the presence of spiked spheric structuresranging in size from 80 to 300 mu which matched the morphologicalcharacteristics of influenza VLPs (FIG. 14B).

Example 4 Purification of Influenza H5 VLPs from Plant Biomass

In addition to an abundant content of soluble proteins, plant leafextracts contain a complex mixture of soluble sugars, nucleic acids andlipids. The crude extract was clarified by a pH shift and heat treatmentfollowed by filtration on diatomaceous earth (see Material and methodsection for a detailed description of the clarification method). FIG.15A (lanes 1-4) presents a Coomassie Blue stained gel comparing proteincontent at the various steps of clarification. A comparison of proteincontent in the crude extract (lane 1) and in the clarified extract (lane4) reveals the capacity of the clarification steps to reduce the globalprotein content and remove most of the major contaminant visible at 50kDa in crude leaf extracts. The 50 kDa band corresponds to the RuBisCOlarge subunit, representing up to 30% of total leaf proteins.

Influenza H5 VLPs were purified from these clarified extracts byaffinity chromatography on a fetuin column. A comparison of the loadfraction (FIG. 15A, lane 5) with the flowthrough (FIG. 15A, lane 6) andthe eluted VLPs (FIG. 15A, lane 7) demonstrates the specificity of thefetuin affinity column for influenza H5 VLPs in plant clarified extract.

The purification procedure resulted in over 75% purity in H5, asdetermined by densitometry on the Coomassie Blue stained SDS-PAGE gel(FIG. 15A, lane 7). In order to assess the structural quality of thepurified product, the purified H5 was concentrated on a 100 NMWL(nominal molecular weight limit) centrifugal filter unit and examinedunder EM after negative staining. FIG. 15B shows a representative sectorshowing the presence of profuse VLPs. A closer examination confirmed thepresence of spikes on the VLPs (FIG. 15C).

As shown in FIG. 15D, H5 VLPs were purified to approx. 89% purity fromclarified leaf extract by affinity chromatography on a fetuin column,based on the density of the Coomassie Blue stained H5 hemagglutinin andon total protein content determination by the BCA method.

The bioactivity of HA VLPs was confirmed by their capacity toagglutinate turkey red blood cells (data not shown).

FIG. 15D also confirms the identity of the purified VLP visualized byWestern blotting and immunodetection with an anti-H5 polyclonal serum(A/Vietnam/1203/2004). A unique band of approximately 72 kDa is detectedand corresponds in size to the uncleaved HA0 form of influenzahemagglutinin. FIG. 15c shows the VLP structure of the vaccine with thehemagglutinin spikes covering its structure.

VLPs were formulated for immunization of mice by filtering through a0.22 filter; endotoxin content was measured using the endotoxin LAL(Limulus Amebocyte Lysate) detection kit (Lonza, Walkserville, Miss.,USA). The filtered vaccine contained 105.8±11.6% EU/ml (endotoxinunits/ml).

Example 5 Localization of Influenza VLPs in Plants

To localize the VLPs and confirm their plasma membrane origin, thin leafsections of H5-producing plants were fixed and examined under TEM afterpositive staining. Observation of leaf cells indicated the presence ofVLPs in extracellular cavities formed by the invagination of the plasmamembrane (FIG. 19). The shape and position of the VLPs observeddemonstrated that despite the apposition of their plasma membranes onthe cell wall, plant cells have the plasticity required to produceinfluenza VLPs derived from their plasma membrane and accumulate them inthe apoplastic space.

Example 6 Plasma Membrane Lipid Analysis

Further confirmation of the composition and origin of the plantinfluenza VLPs was obtained from analyses of the lipid content. Lipidswere extracted from purified VLPs and their composition was compared tothat of highly purified tobacco plasma membranes by high performancethin layer chromatography (HP-TLC). The migration patterns of polar andneutral lipids from VLPs and control plasma membranes were similar.Purified VLPs contained the major phospholipids (phosphatidylcholine andphosphatidylethanolamine) and sphingolipids (glucosyl-ceramide) found inthe plasma membrane (FIG. 27A), and both contained free sterols as thesole neutral lipids (FIG. 27B). However, immunodetection of a plasmamembrane protein marker (ATPase) in purified VLP extracts showed thatthe VLP lipid bilayer does not contain one of the major proteinsassociated with plant plasma membranes, suggesting that host proteinsmay have been excluded from the membranes during the process of VLPsbudding from the plant cells (FIG. 27C).

Example 7 Immunogenicity of the H5 VLPs and Effect of Route ofAdministration

Mice were administered plant-made H5 VLPs by intramuscular injection, orintranasal (inhalation). 0.1 to 12 ug of VLPs were injectedintramuscularly into mice, with alum as an adjuvant, according to thedescribed methods. Peak antibody titers were observed with the lowestantigen quantity, in a similar magnitude to that of 5 ug recombinant,soluble hemagglutinin (H5) (FIG. 20A).

0.1 to 1 ug plant-made H5 VLPs were administered intranasally with achitosan adjuvant provided for an antibody response greater than that ofthe recombinant soluble H5 with an alum adjuvant (FIG. 20B).

For both administration routes, and over a range of antigen quantities,seroconversion was observed in all of the mice tested. Recombinant H5soluble antigen conferred low (<1/40) or negligible (1<1/10 for thenon-adjuvanted recombinant H5) HI titres.

Example 8 Hemagglutination-Inhibition Antibody Titer (HAI) H5 VLP

FIG. 21 A, B illustrates the hemagglutination inhibition (HAI) antibodyresponse 14 days following a “boost” with plant-made H5 VLP, orrecombinant soluble H5. The lowest dose of antigen (0.1 ug) whenadministered intramuscularly produced a superior HAI response to a10-fold greater administration (5 ug) of recombinant soluble H5.Increasing doses of H5 VLP provided a modest increase in HAI over thelowest dose.

HAI response following intranasal administration was significantlyincreased in mice administered plant-made H5 VLPs (1.0 or 0.1 ug)compared to those administered 1 ug recombinant soluble H5, which wassimilar to the negative control. All mice immunized by intramuscularinjection of H5 VLPs (from 0.1 to 12 μg) had higher HAI titers than miceimmunised with the control H5 antigen (FIG. 21A). For the same dose of 5μg, VLPs induced HAI titers 20 times higher than the corresponding doseof the control H5 antigen. VLPs also induced significantly higher HAItiters than the control HA antigen when delivered through the intranasalroute (FIG. 21b ). For a given dose of H5 VLP the levels of HAI titerswere lower in mice immunised intranasally than for mice immunisedintramuscularly; 1 μg VLP induced a mean HAI titer of 210 whenadministered i.m. while the same dose induced a mean HAI titer of 34administered i.n.

When administered intramuscularly, all doses of VLPs induced high levelof antibodies capable of binding homologous whole inactivated viruses(FIGS. 20a and 24). No significant difference was found between theplant-made VLP vaccine and the control H5 antigen (except the 12 μg VLPgroup 14 days after boost), as both antigen preparations induce highbinding antibody titers against the homologous strain. However, whenadministered intranasally, VLPs induced higher binding antibody titersin than did the control H5 antigen (FIG. 20b ). When mixed withChitosan, immunization with one microgram VLP induced a reciprocal meanAb titer of 5 500, 8.6 times higher than the level found in miceimmunized with 1 μg of the control HA antigen (reciprocal mean Ab titerof 920).

The immunogenicity of the plant-derived influenza VLPs was theninvestigated through a dose-ranging study in mice. Groups of five BALB/cmice were immunized intramuscularly twice at 3-week intervals with 0.1μg to 12 μg of VLPs containing HA from influenza A/Indonesia/5/05 (H5N1)formulated in alum (1:1 ratio). Hemagglutination-inhibition titers (HIor HAI), using whole inactivated virus antigen (A/Indonesia/5/05(H5N1)), were measured on sera collected 14 days after the secondimmunization. Immunization with doses of VLP as low as 0.1 μg inducedthe production of antibodies that inhibited viruses from agglutinatingerythrocytes at high dilutions (FIG. 21A). Parallel immunization of micewith 5 μg of non-VLP alum-adjuvanted control H5 antigen (also fromA/Indonesia/5/05) induce an HI response that was 2-3 logs lower thanthat achieved with the lowest VLP dose.

For both administration routes, and over a range of antigen quantities,the HAI response is superior in mice administered VLPs.

Example 9 Effect of Adjuvant on Immunogenicity of H5 VLPs

Plant-made H5 VLPs have a plasma membrane origin (FIG. 19, Example 5).Without wishing to be bound by theory, enveloped viruses or VLPs ofenveloped viruses generally acquire their envelope from the membranethey bud through. Plant plasma membranes have a phytosterol complementthat is rarely, if ever found in animal cells, and several of thesesterols have been demonstrated to exhibit immunostimulatory effects.

Plant-made H5 VLPs were administered intramuscularly (FIG. 22A) orintranasally (FIG. 22B) to mice in the presence or absence of anadjuvant, and the HAI (hemagglutination inhibition antibody response)determined. VLPs, in the presence or absence of an added adjuvant (alumor chitosan, as in these examples) in either system of administrationdemonstrated a significantly greater HAI hemagglutinin inhibition thanrecombinant soluble H5. Even in the absence of an added adjuvant (i.e.alum or chitosan), plant-made H5 VLPs demonstrate a significant HAI,indicative of a systemic immune response to administration of theantigen.

Alum enhanced the mean level of HAI titers by a factor of 5 forintramuscular administration of VLP (FIG. 22a ) and by a factor of 3.7for the control H5 antigen. When administered i.m., 5 μg VLPs induced amean HAI titer 12 times higher than the corresponding dose of control H5antigen. Chitosan did not boost the mean HAI level of the control H5antigen (FIG. 22b ) while it increased the mean HAI level of miceimmunised with 1 μg VLP administered i.n. by a factor of 5-fold.

Example 10 Antibody Isotypes

Mice administered plant-made H5 VLPs or recombinant soluble 115 in thepresence or absence of alum as an added adjuvant demonstrate a varietyof immunoglobulin isotypes (FIG. 23A).

In the presence of an added adjuvant, the antibody isotype profiles ofVLPs and the recombinant H5 are similar, with IgG1 being the dominantisotype. When VLPs or recombinant H5 are administered without an addedadjuvant, IgG1 response is reduced, but remains the dominant isotyperesponse to VLPs, with IgM, IgG2a, IgG2B and IgG3 maintaining similartiters as in the presence of an added adjuvant. IgG1, IgG2a, and IgG2btiters are markedly reduced when recombinant H5 is administered withoutan added adjuvant (FIG. 23A).

These data, therefore, demonstrate that plant-made VLPs do not requirean added adjuvant to elicit a antibody response in a host.

Antibody titers against whole inactivated influenza virus strains(A/Indonesia/5/05; A/Vietnam/1203/04)I in mice administered plant-madeVLPs or soluble recombinant HA intramuscularly in the presence of anadded antigen are illustrated in FIG. 23B. No significant difference isobserved in the antibody titers for these influenza strains in miceadministered 1 ug or 5 ug of VLPs or 5 ug of soluble HA.

Example 11 Cross-Reactivity of Serum Antibodies Induced by the H5 VLPVaccine

Cross-reactivity of serum antibodies induced by H5 VLP was assessedagainst whole inactivated influenza viruses of different strains. AllVLP doses (from 0.1 to 12 μg) as well as 5 μg of control HA antigeninduced high binding antibody titers against a Glade 1 strain(A/Vietnam/1194/04), the homologous strain A/Indonesia/5/05 of Glade2.1, and a Glade 2.2 strain A/turkey/Turkey/1/05 (FIG. 25A).

However, only the plant-made VLP induced HAI titer against theA/turkey/Turkey/1/05 strain (FIG. 25b ). HAI titers for theA/Indonesia/5/05 were high for VLPs.

Example 12 Cross-Protection Conferred by Immunization with Plant-Made H5VLP

Mice that previously had been administered a two-dose regimen ofA/Indonesia/5/05 H5 VLPs as described, were subsequently challengedintranasally with influenza A/Turkey/582/06 (H5N1) (“Turkey H5N1”)infectious virus, and observed. The dose administered, per animal, was10 LD₅₀ (4.09×10⁵ CCID₅₀).

By 7 days post-challenge, only 37.5% of the mice administered the PBSvaccine control had survived exposure to Turkey H5N1 (FIG. 26A). 100% ofanimals administered the control antigen (HA) or 1, 5 or 15 ug ofIndonesia H5 VLPs survived up to 17 days post-challenge, when theexperiment was terminated.

Body mass of the mice was also monitored during the experiment, and theaverage mass of the surviving mice plotted (FIG. 26B). Mice administered1, 5 or 15 ug of the Indonesia H5 VLPs before challenge did not lose anyappreciable mass during the course of the experiment, and in particularmice administered 5 ug of the VLPs appear to have gained significantmass. Negative control mice (no Turkey H5N1 challenge) did notappreciably gain or lose body mass. Positive control mice (notadministered VLPs, but challenged with Turkey H5N1) exhibitedsignificant loss of body mass during the course of the experiment, andthree of these mice died. As body mass is an average of all mice in thecohort, removal of the ‘sickest’ mice (the 3 that died) may lead to anapparent overall increase in mass, however note that the average bodymass of the positive control cohort is still significantly below that ofthe negative or the VLP-treated cohorts.

These data, therefore, demonstrate that plant-made influenza VLPscomprising the H5 hemagglutinin viral protein induce an immune responsespecific for pathogenic influenza strains, and that virus-like particlesmay bud from a plant plasma membrane.

These data, therefore, demonstrate that plants are capable of producinginfluenza virus-like particles, and also for the first time, thatvirus-like particles can bud from a plant plasma membrane.

Further, using the current transient expression technology, a firstantigen lot was produced only 16 days after the sequence of the targetHA was obtained. Under the current yields for H5 VLPs, and at anexemplary dose of 5 μg per subject, each kg of infiltrated leaf mayproduce ˜20,000 vaccine doses. This unique combination of platformsimplicity, surge capacity and powerful immunogenicity provides for,among other embodiments, a new method response in the context of apandemic.

Example 13 Characterization of Hemagglutinin-Containing (H1, H2, H3, H5,H6 and H9) Structures in Plant Extracts Using Size ExclusionChromatography

The assembly of plant-produced influenza hemagglutinin of differentsubtypes into high molecular weight structures was assessed by gelfiltration. Crude or concentrated protein extracts from AGL1/660-,AGL1/540-, AGL1/783-, AGL1/780-, AGL1/785- and AGL1/790-infiltratedplants (1.5 mL) were fractionated by size exclusion chromatography (SEC)on Sephacryl™ S-500 HR columns (GE Healthcare Bio-Science Corp.,Piscataway, N.J., USA). As shown in FIG. 46, Blue Dextran (2 MDa)elution peaked early in fraction 10. When proteins from 200 μL of eachSEC elution fraction were concentrated (5-fold) by acetone-precipitationand analyzed by Western blotting (FIG. 46), hemagglutinins wereprimarily found in fractions 7 to 14, indicating the incorporation of HAinto VLPs. Without wishing to be bound by theory, this suggests that theHA protein had either assembled into a large superstructure or that ithas attached to a high molecular weight structure, irrespectively of thesubtype produced. In FIG. 46, H1 from strain A/New Caledonia/20/1999 andH3 from strain A/Brisbane/10/2007 were produced using PDI signalpeptide-containing cassettes. The results obtained indicate thatreplacement of the native signal peptide by that of alfalfa PDI does notaffect the ability of HA to assemble into particles.

Example 14 Transient Expression of Seasonal Influenza VirusHemagglutinin by Agroinfiltration in N. Benthamiana Plants Using theWild-Type Nucleotide Sequence

The ability of the transient expression system to produce seasonalinfluenza hemagglutinins was determined through the expression of the H1subtype from strains A/Brisbane/59/2007 (H1N1) (plasmid #774), A/NewCaledonia/20/1999 (H1N1) (plasmid #540) and A/Solomon Islands/3/2006(H1N1) (plasmid #775), of the H3 subtype from strains A/Brisbane/10/2007(plasmid #776) and A/Wisconsin/67/2005 (plasmid #777) and of the B typefrom strains B/Malaysia/2506/2004 (Victoria lineage) (plasmid #778) andB/Florida/4/2006 (Yamagata lineage) (plasmid #779). The hemagglutiningene coding sequences were first assembled in the plastocyaninexpression cassette—promoter, 5′UTR, 3′UTR and transcription terminationsequences from the alfalfa plastocyanin gene—and the assembled cassetteswere inserted into to a pCAMBIA binary plasmid. The plasmids were thentransfected into Agrobacterium (AGL1), producing Agrobacterium strainsAGL1/774, AGL1/540, AGL1/775, AGL1/776, AGL1/777, AGL1/778 and AGL1/779,respectively.

N. benthamiana plants were infiltrated with AGL1/774, AGL1/540,AGL1/775, AGL1/776, AGL1/777, AGL1/778 and AGL1/779 and the leaves wereharvested after a six-day incubation period. To determine whether H1accumulated in the agroinfiltrated leaves, protein was first extractedfrom infiltrated leaf tissue and analyzed by Western blotting usinganti-HA antibodies (see Table 6 for the antibodies and conditions usedfor the detection of each HA subtype). For the HA from H1 strains, aunique band of approximately 72 kDa was detected in extracts (FIG. 47),corresponding in size to the uncleaved HA0 form of influenzahemagglutinin. This demonstrated that expression of different annualepidemic strains of hemagglutinin in infiltrated leaves results in theaccumulation of the uncleaved translation product. Using theseexpression and immunodetection strategies, the expression of influenzaHA from H3 subtype or B type was not detected in the crude proteinextracts (FIG. 47).

Example 15 Transient Expression of Potential Pandemic Influenza VirusHemagglutinin by Agroinfiltration in N. Benthamiana Plants Using theWild-Type Nucleotide Sequence

The ability of the transient expression system to produce potentialinfluenza hemagglutinins was determined through the expression of the H5subtype from strains A/Anhui/1/2005 (H5N1) (plasmid #781),A/Indonesia/5/2005 (H5N1) (plasmid #660) and A/Vietnam/1194/2004 (H5N1)(plasmid #782), the H2 subtype from strain A/Singapore/1/1957 (H2N2)(plasmid #780), the H6 from strain A/Teal/Hong Kong/W312/1997 (H6N1)(plasmid #783), the H7 for strain A/Equipe/Prague/1956 (H7N7) (plasmid#784) and finally H9 from strain A/Hong Kong/1073/1999 (H9N2) (plasmid#785). The hemagglutinin gene coding sequences were first assembled inthe plastocyanin expression cassette—promoter, 5′UTR, 3′UTR andtranscription termination sequences from the alfalfa plastocyaningene—and the assembled cassettes were inserted into to a pCAMBIA binaryplasmid. The plasmids were then transfected into Agrobacterium (AGL1),producing Agrobacterium strains AGL1/781, AGL1/660, AGL1/782, AGL1/780,AGL1/783, AGL1/784 and AGL1/785.

N. benthamiana plants were infiltrated with AGL1/781, AGL1/660,AGL1/782, AGL1/780, AGL1/784 and AGL1/785, and the leaves were harvestedafter a six-day incubation period. To determine whether H5 accumulatedin the agroinfiltrated leaves, protein was first extracted frominfiltrated leaf tissue and analyzed by Western blotting usingappropriate anti-HA antibodies (see Table 6 for the antibodies andconditions used for the detection of each HA subtype). A unique band ofapproximately 72 kDa was detected in extracts of plants transformed withH5 and H2 expression constructs (FIGS. 48a and b ), corresponding insize to the uncleaved HA0 form of influenza hemagglutinin. Thisdemonstrated that expression of different potential pandemic strains ofhemagglutinin in infiltrated leaves results in the accumulation of theuncleaved translation product. Using these expression andimmunodetection strategies, the expression of influenza HA from H7 andH9 was not detected in the crude protein extracts (FIG. 48b ).

Example 16 Transient Expression of H5 by Agroinfiltration in N. tabacumPlants

The ability of the transient expression system to produce influenzahemagglutinin in leaves of Nicotiana tabacum was analysed through theexpression of the H5 subtype from strain A/Indonesia/5/2005 (H5N1)(plasmid #660). The hemagglutinin gene coding sequences were firstassembled in the plastocyanin expression cassette—promoter, 5′UTR, 3′UTRand transcription termination sequences from the alfalfa plastocyaningene—and the assembled cassettes were inserted into to a pCAMBIA binaryplasmid. The plasmids was then transfected into Agrobacterium (AGL1),producing strain AGL1/660.

N. tabacum plants were infiltrated with AGL1/660 and the leaves wereharvested after a six-day incubation period. To determine whether H5accumulated in the agroinfiltrated leaves, proteins were first extractedfrom infiltrated leaves and analyzed by Western blot using anti-H5antibodies. A unique band of approximately 72 kDa was detected inextracts (FIG. 49), corresponding in size to the uncleaved HA0 form ofinfluenza hemagglutinin. This demonstrated that expression ofhemagglutinin in infiltrated N. tabacum leaves results in theaccumulation of the uncleaved HA0 precursor.

Example 17 Immunogenicity of Plant-Made H5N1 VLP Vaccine fromA/Indonesia/5/05 (H5N1) in Ferrets

A dose escalation study in ferrets was performed to evaluate theimmunogenicity of plant derived VLPs. In vitro cross-reactivity of serumantibody induced by the 115 VLP vaccine at 3 doses (1, 5 and 15 ug) wasassessed by hemagglutination inhibition of three other H5N1strains—A/turkey/Turkey/1/05 (clade 2.2), A/Vietnam/1194/04 (clade 1)and A/Anhui/5/05 (all whole, inactivated virus), using serum taken 14days after the first dose of vaccine (FIG. 50A), and 14 days after the2^(nd) dose (FIG. 50 B). For all 3 dose concentrations, cross-reactivityis observed

Example 18 Analysis of the Immunogenicity Results According to CHMPCriteria

The EMEA's Committee for Medicinal Products for Human Use (CHMP)(http://www.emea.europa.eu/htms/general/contacts/CHMP/CHMP.html) setsout three criteria (applied following the second dose) for vaccineefficacy: 1—Number of seroconversion or significant increase in HItiters (4-fold)>40%; 2—Mean geometric increase of at least 2.5;3—proportion of subjects achieving an HI titer of 1/40 should be atleast 70%. Analysis of these criteria in the ferret model is shown inTables 8-11. (*) is indicative of meeting or exceeding the CHMPcriteria. A summary of cross-immunogenicity analysis in relation to CHMPcriteria for licensure is shown in Table 12.

Animals were assessed daily for body weight, temperature and overallcondition. No sign of sickness or discomfort was recorded during thestudy. Body weight and temperature was within normal ranges during thestudy. The vaccine was safe and tolerated by the animals.

TABLE 8 Data for homologous strain (A/Indonesia/5/05) Study group 1 μg 5μg 15 μg 5 μg Day Criteria 1 μg adjuvanted 5 μg adjuvanted 7.5 μg 15 μgadjuvanted 30 μg ITC 14 (post % 4-fold increase in HI titer 0% 100%  0%100% * 20%  20%  80% * 0% 0% 1st inj.) Mean geometric increase 0%   7.60% 15.6 * 1.3 1.2   11.2 * 0% 0% % of HI titer of 1/40 0% 60% 0% 100% *20%  0% 80% * 0% 0% Mean HI titer 38 78   56 35 (14 % 4-fold increase inHI titer 0%  100% * 0%  60% * 0% 0% 40% * 0% 0% days post Mean geometricincrease 0%   10.8 * 0%  5.9 * 0.7 0%   4 * 0% 0% boost) % of HI titerof 1/40 0%  100% * 0% 100% * 0% 0% 100% *  0% 0% Mean HI titer 411 465   217 

TABLE 9 Data for heterologous strain (A/Vietnam/1194/04) Study group 1μg 5 μg 15 μg 5 μg Day Criteria 1 μg adjuvanted 5 μg adjuvanted 7.5 μg15 μg adjuvanted 30 μg ITC 14 (post % 4-fold increase in HI titer 0% 0%  0% 1st inj.) Mean geometric increase 1.2 1.2 1.3  % of HI titer of 1/400% 0%   0% 35 (post % 4-fold increase in HI titer 60%  80% * 60% boost)Mean geometric increase 2.3   5.1 * 1.78 % of HI titer of 1/40 0% 80% *20%

TABLE 10 Data for heterologous strain (A/turkey/Turkey/1/05) Study group1 μg 5 μg 15 μg 5 μg Day Criteria 1 μg adjuvanted 5 μg adjuvanted 7.5 μg15 μg adjuvanted 30 μg ITC 14 (post % 4-fold increase in HI titer 40% 20%  60%  1st inj.) Mean geometric increase 1.9  1.7  2.8   % of HItiter of 1/40 40%  20%  40%  35 (post % 4-fold increase in HI titer 80% * 100% *  80% * boost) Mean geometric increase 10.6 * 20.8 * 7.7 *% of HI titer of 1/40 100% * 100% * 100% *

TABLE 11 Data for heterologous strain (A/Anhui/5/05) Study group 1 μg 5μg 15 μg 5 μg Day Criteria 1 μg adjuvanted 5 μg adjuvanted 7.5 μg 15 μgadjuvanted 30 μg ITC 14 (post % 4-fold increase in HI titer 40%  20% 80% * 1st inj.) Mean geometric increase 1.8  1.3  6.4 * % of HI titer of1/40 20%  20%  80% * 35 (post % 4-fold increase in HI titer 100% *100% * 60% * boost) Mean geometric increase 11.8 * 14.4 * 3 *   % of HItiter of 1/40 100% *  80% * 80% *

TABLE 12 Summary of cross-immunogenicity analysis in relation to CHMPcriteria for licensure. Study group 1 μg 5 μg 15 μg adju- adju- adju-Strain Criteria vanted vanted vanted A/turkey/ % 4-fold increase in HItiter  80% * 100% * 80% * Turkey/1/05 Mean geometric increase 10.6 *20.8 * 7.7 * (clade 2.2 % of HI titer of 1/40 100% * 100% * 100% * A/Anhui/1/ % 4-fold increase in HI titer 100% * 100% * 60% * 05 (cladeMean geometric increase 11.8 * 14.4 * 3 *  2.3) % of HI titer of 1/40100% *  80% * 80% * A/Vietnam/ % 4-fold increase in HI titer 60%   80% *60%  1194/04 Mean geometric increase 2.3   7.1 * 1.78  (clade 1) % of HItiter of 1/40  0%  80% * 20% 

Example 19 Selection of Hemagglutinin Nucleotide Sequences

The nucleotide sequences of the HA were retrieved from an influenzasequence database (see URL: flu.lanl.gov), or the NCBI influenza virusresource (Bao et al., 2008. J. Virology 82(2): 596-601; see URL:ncbi.nlm.nih.gov/genomes/FLU/FLU.html). For several of the HA nucleicacid sequences, multiple entries are listed in the databases (Table 13).Some variation is associated primarily with the culture system(Origin—MDCK, egg, unknown, viral RNA/clinical isolate); for example,the glycosylation site at position 194 (mature protein numbering) of theHA is absent when type B influenza virus is expressed in allantoic fluidof eggs (see also Chen et al., 2008). For some sequences, domains may belacking (e.g. incomplete clones, sequencing artifacts, etc.). Domainsand sub-domains of influenza hemagglutinin are discussed generally inthe Description. Domains or subdomains of a first sequence may becombined with a domain from a second existing sequence e.g. the signalpeptide of a first strain sequence may be combined with the balance ofthe hemagglutinin coding sequence from a second strain to provide acomplete coding sequence.

TABLE 13 Variation in Influenza subtypes for selected HA codingsequences Sequence database Strain reference No. Origin SP HA1 HA2 DTmDivergence H1 A/Solomon ISDN231558 MDCK Y Y Y Y 189: R ou G, 220: K(MDCK) Islands/3/2006 (Vaccine rec.) T(Egg), 249: Q (MDCK) R(Egg), 550:L (MDCK) R (Egg) A/Solomon ISDN238190 Egg Y Y Y Y 189: R ou G, 220: K(MDCK) Islands/3/2006 T(Egg), 249: Q (MDCK) R(Egg), 550: L (MDCK) R(Egg) A/Solomon EU100724 ? Y Y Y Y 189: R ou G, 220: K (MDCK)Islands/3/2006 T(Egg), 249: Q (MDCK) R(Egg), 550: L (MDCK) R (Egg)A/Solomon ISDN220951 MDCK Y Y N N 189: R ou G, 220: K (MDCK)Islands/3/2006 T(Egg), 249: Q (MDCK) R(Egg), 550: L (MDCK) R (Egg)A/Solomon ISDN220953 Egg Y Y N N 189: R ou G, 220: K (MDCK)Islands/3/2006 T(Egg), 249: Q (MDCK) R(Egg), 550: L (MDCK) R (Egg)A/Solomon EU124137 Egg Y Y N N 189: R ou G, 220: K(MDCK) Islands/3/2006T(Egg), 249: Q (MDCK) R(Egg), 550: L (MDCK) R (Egg) A/Solomon EU124135MDCK Y Y N N 189: R ou G, 220: K (MDCK) Islands/3/2006 T(Egg), 249: Q(MDCK) R(Egg), 550: L (MDCK) R (Egg) A/Solomon EU124177 MDCK Y Y Y Y189: R ou G, 220: K (MDCK) Islands/3/2006 T(Egg), 249: Q (MDCK) R(Egg),550: L (MDCK) R (Egg) H1 A/Brisbane/ ISDN282676 MDCK Y Y Y 203: D/I/N Dest le plus 59/2007 abondant chez les H1 A/Brisbane/ ISDN285101 Egg Y YN N 203: D/I/N D est le plus 59/2007 abondant chez les H1 A/Brisbane/ISDN285777 Egg Y Y Y Y 203: D/I/N D est le plus 59/2007 abondant chezles H1 A/Brisbane/ ISDN282677 Egg Y Y Y Y 203: D/I/N D est le plus59/2007 abondant chez les H1 H3 A/Brisbane/ ISDN274893 Egg Y Y Y Y 202:V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/I A/Brisbane/ ISDN257648MDCK N Y Y Y 202: V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/IA/Brisbane/ ISDN256751 Egg Y Y Y Y 202: V/G, 210: L/P, 215: del 10/2007Ala, 242: S/I A/Brisbane/ ISDN273757 Egg Y Y Y Y 202: V/G, 210: L/P,215: del 10/2007 Ala, 242: S/I A/Brisbane/ ISDN273759 Egg Y Y Y Y 202:V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/I A/Brisbane/ EU199248 Egg NY Y Y 202: V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/I A/Brisbane/EU199366 Egg Y Y Y Y 202: V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/IA/Brisbane/ ISDN257043 Egg N Y Y Y 202: V/G, 210: L/P, 215: del 10/2007Ala, 242: S/I A/Brisbane/ EU199250 MDCK N Y Y Y 202: V/G, 210: L/P, 215:del 10/2007 Ala, 242: S/I A/Brisbane/ ISDN275357 Egg N Y N N 202: V/G,210: L/P, 215: del 10/2007 Ala, 242: S/I A/Brisbane/ ISDN260430 Egg N YY Y 202: V/G, 210: L/P, 215: del 10/2007 Ala, 242: S/I H3 A/Wisconsin/ISDN131464 ? N Y Y N 138: A/S 67/2005 (vaccine rec.) 156: H/Q 186: G/V196: H/Y A/Wisconsin/ DQ865947 ? N Y partiel N 138: A/S 67/2005 156: H/Q186: G/V 196: H/Y A/Wisconsin/ EF473424 ? N Y Y N 138: A/S 67/2005 156:H/Q 186: G/V 196: H/Y A/Wisconsin/ ISDN138723 Egg N Y Y Y 138: A/S67/2005 156: H/Q 186: G/V 196: H/Y A/Wisconsin/ EF473455 Egg N Y Y Y138: A/S 67/2005 156: H/Q 186: G/V 196: H/Y A/Wisconsin/ ISDN138724 ? NY Y Y 138: A/S 67/2005 156: H/Q 186: G/V 196: H/Y B B/Malaysia/ISDN126672 Egg Y Y N N 120 K/N 2506/2004 (vaccine rec.) 210 T/AB/Malaysia/ EF566433 Egg Y Y N N 120 K/N 2506/2004 210 T/A B/Malaysia/ISDN231265 Egg Y Y Y Y 120 K/N 2506/2004 210 T/A B/Malaysia/ ISDN231557MDCK Y Y Y Y 120 K/N 2506/2004 210 T/A B/Malaysia/ EF566394 MDCK Y Y N N120 K/N 2506/2004 210 T/A B/Malaysia/ EU124274 Egg Y Y Y Y 120 K/N2506/2004 210 T/A B/Malaysia/ EU124275 MDCK Y Y Y Y 120 K/N 2506/2004210 T/A B/Malaysia/ ISDN124776 MDCK Y Y N N 120 K/N 2506/2004 210 T/A BB/Florida/4/ ISDN261649 Egg Y Y Y N lacking glycosylation site at 2006position 211; 10 amino acids of DTm/cytoplasmic tail B/Florida/4/EU100604 MDCK N Y N N 2006 B/Florida/4/ ISDN218061 MDCK N Y N N 2006B/Florida/4/ ISDN285778 Egg Y Y Y Y Includes cytoplasmic tail 2006 BB/Brisbane/3/ ISDN256628 Egg N Y N N lacking glycosylation site at 2007position 211 B/Brisbane/3/ ISDN263782 Egg Y Y Y Y lacking glycosylationsite at 2007 position 211 B/Brisbane/3/ ISDN263783 MDCK Y Y Y Y 2007 H5A/Viet Nam/ ISDN38686 ? Y Y Y Y 1194/2004 (Vaccine rec.) A/Viet Nam/AY651333 ? Y Y Y Y 1194/2004 A/Viet Nam/ EF541402 ? Y Y Y Y 1194/2004 H5A/Anhui1/1/ DQ37928 ? Y Y Y Y 2005 (vaccine rec.) A/Anhui1/1/ ISDN131465Egg Y Y Y Y 2005 H7 A/Chicken/Italy/ AJ91720 ARN Y Y Y Y 13474/1999 genH7 A/Equine/ AB298277 ? Y Y Y Y 152 (R/G) Prague/56 (Lab 169 (T/I)reassortant) 208 (N/D) (glycosylation site abolished) A/Equine/ X62552 ?Y Y Y Y Prague/56 H9 A/Hong Kong/ AJ404626 ? Y Y Y Y 1073/1999 A/HongKong/ AB080226 ? N Y N N 1073/1999 H2 A/Singapore/ AB296074 ? Y Y Y Y1/1957 A/Singapore/ L20410 RNA Y Y Y Y 1/1957 A/Singapore/ L11142 ? Y YY Y 1/1957 H2 A/Japan/305/ L20406 ? Y Y Y Y 1957 A/Japan/305/ L20407 ? YY Y Y 1957 A/Japan/305/ CY014976 ? Y Y Y Y 1957 A/Japan/305/ AY209953 ?Y Y N N 1957 A/Japan/305/ J02127 ? Y Y Y Y 1957 A/Japan/305/ DQ508841 ?Y Y Y Y 1957 A/Japan/305/ AY643086 ? Y Y Y N 1957 A/Japan/305/ AB289337? Y Y Y Y 1957 A/Japan/305/ AY643085 ? Y Y Y Y 1957 A/Japan/305/AY643087 Drug Y Y Y N 1957 resistant H6 A/Teal/Hong Kong/ AF250479 Egg YY Y Y W312/1997 (H6N1) Y, N—Yes, No, respectively SP—presence of signalpeptide sequence Y/N HA1—complete HA1 domain Y/N HA2—complete HA2 domainY/N DTm—complete transmembrane domain Y/NStrain: H1 from A/Solomon Islands/3/2006

Eight amino acid sequences were compared, and variations identified.(Table 14). Position 171 exhibited a variation of glycine (G) orarginine (R) in some sequences.

TABLE 14 A/Solomon Islands/3/2006 amino acid variation Amino acid # MDCKEgg 212 K T 241 Q R 542 L R Numbering from the starting MStrain: H1 from A/Brisbane/59/2007

Position 203 exhibited a variation of aspartic acid (D), isoleucine (I)or asparagine (N).

Strain: H3 from A/Brisbane/10/2007

Sequence variations were observed at 5 positions (Table 15). In position215, a deletion is observed in two sampled sequences.

TABLE 15 H3 from A/Brisbane/10/2007 amino acid variation Origin 202,210, 215, 235 242* ISDN274893 Egg V L — Y I ISDN273759 Egg G P A S IEU199248 Egg G P A S I EU199366 Egg G P A S I ISDN273757 Egg V L — S SISDN257043 Egg G P A S I EU199250 MDCK G L A S I ISDN375357 Egg G P A SI ISDN260430 Egg G P A S I ISDN256751 Egg G P A S I ISDN257648 MDCK G LA S I *Numbering from the starting MStrain: H3 from A/Wisconsin/67/2005

Sequence variations in this strain were observed at 4 positions (Table16).

TABLE 16 H3 from A/Wisconsin/67/2005 amino acid variation Origin 138,156, 186, 196 ISDN138724 Unknown A H G H DQ865947 Unknown S H V YEF473424 Unknown A H G H ISDN138723 Egg S Q V Y ISDN131464 Unknown A H GH EF473455 Egg A H G H *Numbering from the mature proteinStrain: B from B/Malaysia/2506/2004

Variation at two positions is observed (Table 17). Position 120 is not aglycosylation site; position 210 is involved in glycosylation; thisglycosylation is abolished following culture in eggs.

TABLE 17 Hemagglutinin from B/Malaysia/2506/2004 amino acid variationAmino acid # MDCK Egg 120 K N 210 T A * Numbering from the middle of SPStrain: hemagglutinin from B/Florida/4/2006; ISDN261649

Observed variations include amino acid sequence variation at position211, depending on the culture system. Asparatine (N) is found insequences isolated from MDCK cells, while glutamic acid (D) is found insequence isolated from eggs. Position 211 is a glycosylation site, andis abolished following culture in eggs.

Strain: H2 from A/Singapore/1/1957

Sequence variations were observed in 6 positions (Table 18).

TABLE 18 H2 from A/Singapore/1/1957 amino acid variation Ammo acid No.Origin 166 168 199\236 238 358 L20410 Viral RNA K E T L S V L11142Unknown E G K L S I AB296074 Unknown K G T Q G V Consensus K G T Q/L G VA/Japan/305/1957 ¹Numbering from the mature proteinStrains: H5 from A/Vietnam/1194/2004 and H5 from A/Anhui/1/2005

There were no variations observed in the amino acid sequence uponaligning the primary sequences of either of these H5 strains.

Strain: H6 from A/Teal/Hong Kong/W312/1997

Only one entry was available for strain (AF250179).

Strain: H7 from A/Equine/Prague/56

A total of 2 sequence entries were found in the databases. The entryAB298877 was excluded as it is a laboratory reassortant.

Strain: H9 from A/Hong Kong/1073/1999; AJ404626

A total of 2 sequence entries were found in the databases. Only one wascomplete.

Example 20 Transient Expression of Influenza Virus Hemagglutinin Fusedto a Signal Peptide from a Plant Secreted Protein

The effect of signal peptide modification on HA accumulation level forother hemagglutinins was also investigated through the expression of theA subtype HAs from strains A/Brisbane/59/2007 (H1N1) (plasmid #787),A/New Caledonia/20/1999 (H1N1) (plasmid #540), from strainsA/Brisbane/10/2007 (H3N2) (plasmid 790) and A/Indonesia/5/2005 (H5N1)(plasmid #663) and of the B type from strains B/Florida/4/2006 (plasmid#798) fused to the signal peptide (SP; nucleotides 32-103) from ofalfalfa protein disulfide isomerase (PDI; accession No. Z11499; SEQ. ID.NO: 34; FIG. 17). The PDI SP-hemagglutinin gene fusions were assembledin the plastocyanin expression cassette—promoter, 5′UTR, 3′UTR andtranscription termination sequences from the alfalfa plastocyaningene—and the assembled cassettes were inserted into to a pCAMBIA binaryplasmid. The plasmids were then transfected into Agrobacterium (AGL1),producing Agrobacterium strains AGL1/787, AGL1/540, AGL1/790, AGL1/663and AGL1/798, respectively.

N. benthamiana plants were infiltrated with AGL1/787, AGL1/540,AGL1/790, AGL1/663 and AGL1/798. In parallel, a series of plants wasinfiltrated with AGL1/774, AGL776, AGL1/660 and AGL1/779 for comparisonpurposes. Leaves were harvested after a six-day incubation period andproteins were extracted from infiltrated leaves and analyzed by Westernblot using the appropriate anti-HA antibodies. The expression of HA fromH1/Brisbane and H3/Brisbane were considerably improved using the SP fromPDI compared to the expression observed for the same HAs with theirnative signal peptide (FIGS. 87b and c , respectively). The expressionof a third HA from subtype H1 (strain A/New Caledonia/20/1999) wasconfirmed using this SP replacement strategy (FIG. 87a ). Themodification of sognal peptide did not lead to substantial increase inHA accumulation for H5 (A/Indonesia/5/2005) (FIG. 87d ), and no signalwas detected for HA from strain B/Florida/4/2006, irrespectively of thesignal peptide used for expression (FIG. 87e ). For all the conditionswhere the expression of HA was detected, a unique immunoreactive bandwas observed at a molecular weight of approximately 72 kDa (FIG. 87a tod ), corresponding in size to the uncleaved HA0 precursor.

Example 21 HA Expression Under the Control of CPMV-HT ExpressionCassette

An expression cassette CPMV-HT (Sainsbury et al. 2008 Plant Physiology148: 1212-1218; see also WO 2007/135480) comprising untranslatedsequences from the Cowpea mosaic virus (CPMV) RNA2 was used forexpression of some hemagglutinins in transgenic plants. HA from A/NewCaledonia/20/1999 (H1), A/Brisbane/59/2007 (H1), A/Brisbane/10/2007(H3), A/Indonesia/5/2005 (H5) and B/Florida/4/2006 (B) were expressedunder the control of CPMV-HT in N. benthamiana plants, agroinfiltratedas described. After incubation, leaves were harvest, extracted and HAcontents in protein extracts were compared by Western blot. As shown inFIG. 88, the CPMV-HT expression cassette led to higher HA expressionlevel than the plastocyanin cassette, irrespectively of the signalpeptide used. Furthermore, for strain B from B/Florida/4/2006, the useof CPMV-HT expression cassette allowed the detection of HA accumulationwhich remained undetectable under these immunodetection conditions whenexpressed under the plastocyanin cassette.

TABLE 19 Expression cassette used for expression of influenzahemagglutinins with native or PDI signal peptides. Agro SignalExpression strain HA expressed Peptide Cassette AGL1/540 H1 (A/NewCaledonia/20/99) PDI Plastocyanin AGL1/580 H1 (A/New Caledonia/20/99)PDI CPMV-HT AGL1/774 H1 (A/Brisbane/59/2007) native PlastocyaninAGL1/787 H1 (A/Brisbane/59/2007) PDI Plastocyanin AGL1/732 H1(A/Brisbane/59/2007) native CPMV-HT AGL1/776 H3 (A/Brisbane/10/2007)native Plastocyanin AGL1/790 H3 (A/Brisbane/10/2007) PDI PlastocyaninAGL1/735 H3 (A/Brisbane/10/2007) native CPMV-HT AGL1/736 H3(A/Brisbane/10/2007) PDI CPMV-HT AGL1/660 H5 (A/Indonesia/5/2005) nativePlastocyanin AGL1/685 H5 (A/Indonesia/5/2005) native CPMV-HT AGL1/779 B(B/Florida/4/2006) native Plastocyanin AGL1/798 B (B/Florida/4/2006) PDIPlastocyanin AGL1/738 B (B/Florida/4/2006) native CPMV-HT AGL1/739 B(B/Florida/4/2006) PDI CPMV-HT

Example 22 Co-Expression with Hsp70 and Hsp40 in Combination with SignalPeptide Modification

Cytosolic Hsp70 and Hsp40 (construct number R870) of plant origin wereco-expressed with H1 New Caledonia (construct number 540) or H3 Brisbane(construct number 790), both bearing a signal peptide of plant origin(alfalfa PDI signal peptide). The co-expression was performed byagroinfiltration of N. benthamiana plants with a bacterial suspensioncontaining a mixture (1:1:1 ratio) of AGL1/540, AGL1/R870, AGL1/35SHcPro(For H1) or AGL1/790, AGL1/R870 and AGL1/35SHcPro (for H3). Controlplants were agroinfiltrated with a mixture (1:2 ratio) of AGL1/540,AGL1/35SHcPro (for H1) or AGL1/790, AGL1/35SHcPro (for H3). Afterincubation, leaves were harvest, extracted and HA contents in proteinextracts were compared by Western blot (FIG. 89). In the conditionstested the results obtained indicate that the co-expression of Hsp70 andHsp40 did not increase hemagglutinin accumulation level for H1 NewCaledonia. However, for H3 Brisbane, the Western blot clearly indicatedthat the co-expression of cytosolic Hsp70 and Hsp40 resulted in asignificant increase in hemagglutinin accumulation level.

All citations are hereby incorporated by reference.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

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What is claimed is:
 1. A virus like particle (VLP) comprising protein,and one or more than one lipid derived from a plant, the proteinconsisting of influenza hemagglutinin (HA), and the VLP beingcharacterized as having a size of from 80 to 300 nm.
 2. A compositioncomprising an effective dose of the VLP of claim 1 for inducing immunityto an influenza virus in a subject, and a pharmaceutically acceptablecarrier.
 3. The composition of claim 2, wherein the virus like particleis prepared for administration to the subject orally, intradermally,intranasally, intramuscularly, intraperitoneally, intravenously, orsubcutaneously.
 4. The virus like particle (VLP) of claim 1, wherein theinfluenza HA protein comprises one or more than one plant-specificN-glycans, or modified N-glycans.
 5. A composition comprising aneffective dose of the VLP of claim 4 for inducing immunity to aninfluenza virus in a subject, and a pharmaceutically acceptable carrier.6. The composition of claim 5, wherein the virus like particle isprepared for administration to the subject orally, intradermally,intranasally, intramuscularly, intraperitoneally, intravenously, orsubcutaneously.